WO2015143006A1 - Engineered chimeric pegylated adi and methods of use - Google Patents

Engineered chimeric pegylated adi and methods of use Download PDF

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Publication number
WO2015143006A1
WO2015143006A1 PCT/US2015/021189 US2015021189W WO2015143006A1 WO 2015143006 A1 WO2015143006 A1 WO 2015143006A1 US 2015021189 W US2015021189 W US 2015021189W WO 2015143006 A1 WO2015143006 A1 WO 2015143006A1
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WIPO (PCT)
Prior art keywords
microorganism
mycoplasma
adi
recombinant chimeric
peg
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PCT/US2015/021189
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French (fr)
Inventor
Bor-Wen WU
Robert Almassy
Wei He
Richard E. Showalter
Jiaojuan HE
Yunyun GUO
James A. Thomson
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Tdw Group
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Application filed by Tdw Group filed Critical Tdw Group
Priority to KR1020167028878A priority Critical patent/KR102353046B1/en
Priority to EP15765975.6A priority patent/EP3119421B1/en
Priority to JP2017501094A priority patent/JP6612316B2/en
Priority to US15/125,833 priority patent/US10463721B2/en
Priority to SG11201607736UA priority patent/SG11201607736UA/en
Priority to KR1020227001440A priority patent/KR102523031B1/en
Priority to CN201580022377.XA priority patent/CN106794229B/en
Publication of WO2015143006A1 publication Critical patent/WO2015143006A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03006—Arginine deiminase (3.5.3.6)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to engineered ADI, in particular recombinant chimeric ADI proteins engineered to reduce antigenicity.
  • engineered chimeric ADI proteins are useful for treating arginine-dependent diseases such as cancer.
  • Amino acid deprivation therapy can be an effective treatment of some forms of cancer.
  • This treatment is particuiariy effective for acute lymphoblastic leukemia (Avramis 2005, Viera Pinheiro 2004).
  • Acute lymphoblastic leukemia cells require the amino acid asparagine for growth and proliferation.
  • most normal human cells are capable of synthesizing asparagine and are unaffected by asparagine depletion. Therefore, decreasing serum asparagine with asparaginase can selectively kill the cancer ceils without harming the normal cells, tissues, and host.
  • Arginine is another non-essentiai amino acid for humans and mice (for review see Rogers 1994).
  • arginine can be synthesized from citruiline in two steps via the Krebs (urea) cycle enzymes argininosuccinate synthetase (ASS, L-citru!iine:L-aspartate iigase [AMP-forming], EC 8.3.4.5) and argininosuccinate lyase (ASL, L-argininosuccinate arginine-!yase, EC 4.3.2.) (Haines 201 1 , Wu 2009, Morris 2006, Husson 2003, Tapiero 2002, Rogers 1994).
  • ASS catalyzes the conversion of citruiline and aspartic acid to argininosuccinate, which is then converted to arginine and fumaric acid by ASL.
  • ADi-PEG 20 treatment requires multiple doses over a period of time. After a number of treatments, anti-ADI-PEG 20 antibodies can develop that may limit its continued effectiveness. Therefore, there is a need in the art for ADI that is engineered to improve and extend the efficacy of arginine depletion therapy.
  • One aspect of the present invention provides a recombinant chimeric arginine deiminase (ADI) comprising a catalytic domain of an ADI protein derived from a first microorganism and an a-helical domain of an ADI protein derived from a second
  • the first microorganism is selected from the genera Mycoplasma, Clostridium, Bacillus, Borrelia, Enterococcus, Streptococcus, Lactobacillus, and Giardia, In a further embodiment, the first microorganism is selected from the group consisting of Mycoplasma pneumonia, Mycoplasma hominis, Mycoplasma arginini,
  • the first microorganism is selected from the group consisting of M. arginini, M. arthritidis, M. hominis, Mycoplasma pneumonia, Mycoplasma phocicerebrale, Mycoplasma orale, Mycoplasma galeae,
  • Mycoplasma phocidae Mycoplasma columbinum, Mycoplasma iowae, Mycoplasma crocodyli, Mycoplasma fermentans, Mycoplasma gaiiinarum, Mycoplasma iners,
  • the second microorganism optionally differs from the first microorganism and is selected from the genera Mycoplasma, Clostridium, Bacillus, Borreiia, Enterococcus, Streptococcus, Lactobacillus, and Giardia. In another embodiment, the second microorganism optionally differs from the first microorganism and is selected from the genera Mycoplasma, Clostridium, Bacillus, Borreiia, Enterococcus, Streptococcus, Lactobacillus, and Giardia. In another embodiment, the second microorganism optionally differs from the first microorganism and is selected from the genera Mycoplasma, Clostridium, Bacillus, Borreiia, Enterococcus, Streptococcus, Lactobacillus, and Giardia. In another embodiment, the second microorganism optionally differs from the first microorganism and is selected from the genera Mycoplasma, Clostridium, Bacillus, Bor
  • the microorganism is selected from the group consisting of Mycoplasma pneumonia, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Streptococcus pyogenes, streptococcus pneumonia, Borreiia burgdorferi, Borreiia afzelii, Giardia intestinails, Clostridium perfnngens, Bacillus licneniformis, and Enterococcus faecaiis.
  • the second microorganism optionally differs from the first
  • microorganism and is selected from the group consisting of M. arginini, M. arthritidis, M, hominis, Mycoplasma pneumonia, Mycoplasma phocicerebrale, Mycoplasma orale,
  • Mycoplasma gateae Mycoplasma phocidae, Mycoplasma columbinum, Mycoplasma lowae, Mycoplasma crocodyli, Mycoplasma fermentans, Mycoplasma gaiiinarum, Mycoplasma iners, Mycoplasma penetrans, Mycoplasma gallisepticum, Mycoplasma alligatoris,
  • Mycoplasma mobile and Mycoplasma capricolum.
  • the first microorganism is selected from the group consisting of Mycoplasma gaiiinarum, Mycoplasma iners, and Mycoplasma columbinum and the second microorganism is selected from the group consisting of Mycoplasma gaiiinarum, Mycoplasma iners, and Mycoplasma columbinum, wherein the first and second
  • microorganism are optionally different microorganisms.
  • the first microorganism is M. arginini and the second microorganism is M. arthritidis, and in other specific embodiments, the first microorganism is M. arginini and the second microorganism is M. hominis or the first microorganism is M. arthritidis and the second microorganism is M. arginini. in certain embodiments, the first microorganism is M. gateae and the second microorganism is M. arthritidis. in certain embodiments, the first microorganism is M. gateae and the second microorganism is M. columbinum. In some embodiments, the first microorganism is M.
  • the first microorganism is M. gateae and the second microorganism is M. phocicerebrale.
  • the first microorganism is M. gateae and the second microorganism is M. phocidae.
  • the first microorganism is M. phocicerebrale and the second microorganism is M. arginini.
  • the first microorganism is M. phocicerebrale and the second microorganism is M. gateae. in specific embodiments, the first microorganism is M. phocicerebrale and the second microorganism is M. phocicerebrale.
  • the first microorganism is M.
  • the first microorganism is M. phocidae and the second microorganism is M. arginini.
  • the first microorganism is M. phocidae and the second microorganism is M. arthritidis.
  • the first microorganism is M. phocidae and the second microorganism is M. coiumhinum.
  • the first microorganism is M. phocidae and the second microorganism is M. gateae.
  • the first microorganism is M. phocidae and the second microorganism is M. phocicerebrale.
  • the first microorganism is M. gal!inarum and the second microorganism is M. coiumhinum.
  • the first microorganism is M, gaiiinarum and the second
  • microorganism is M. iners
  • first microorganism is M. iners and the second microorganism is M. columbinum.
  • first microorganism is iners and the second microorganism is M. gaiiinarum.
  • illustrative recombinant chimeric ADI molecules comprise, consist, or consist essentially of the amino acid sequence set forth in any one of SEQ ID NOs:4-13 or 22-59 or a variant thereof having at least 80% or 90% sequence identity to any of SEQ ID NOs:4-13 or 22-59.
  • the recombinant chimeric ADi has been modified to remove at least one pegylation site.
  • at least one lysine residue has been modified by an amino acid substitution.
  • at least 5 lysine residues have been modified by an amino acid substitution
  • at least 10 lysine residues have been modified by an amino acid substitution
  • at least 15 lysine residues have been modified by an amino acid substitution
  • at least 20 lysine residues have been modified by an amino acid substitution.
  • Illustrative recombinant chimeric ADI molecules as described herein comprise the amino acid sequence set forth in any one of SEQ I D NOs: 10-13.
  • the ADI is covalently bonded via a biocompatible linker to polyethylene glycol.
  • the arginine deiminase may be covalently bonded to more than one polyethylene glycol molecule and in certain embodiments may be covalently bonded to about 1 to about 10 polyethylene glycol molecules and in one specific embodiment, to 5 ⁇ 3 PEG molecules.
  • the PEG molecules covalently bonded to the ADI as described herein may be straight chain or branch chain PEG molecules and may have a total weight average molecular weight of from about 1 ,000 to about 40,000 and in one embodiment, from about 10,000 to about 30,000. in certain embodiments of the recombinant chimeric AD!
  • the biocompatible linker comprises a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a hisfidine group, a methylene group, or any combinations thereof.
  • the source of the succinyl group is succinimidyl succinate.
  • aspects of the invention provide a polynucleotide encoding the recombinant chimeric AD I described herein, vectors comprising the polynucleotide and isolated host cells comprising such vectors.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences,
  • Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate.
  • plasmids viral e.g. phage, or phagemid, as appropriate.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • polynucleotides are commonly referred to as "codon-optimized.” Any of the polynucleotides described herein may be utilized in a codon-optimized form. In certain embodiments, a polynucleotide can be codon optimized for use in specific bacteria such as E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown ef a/., Protein Expr Purif. 59:94-102, 2008).
  • Suitable host cells include mammalian cells, bacteria, yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, HEK-293 cells, human fibrosarcoma cell line HT-1080 (see, e.g.,
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney ceils (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney ceils (MDCK, ATCC CCL 34); buffalo rat liver ceils (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TR1 cells (Mather ef ai, Annals N. Y. Acad. Sci.
  • MRC 5 cells MRC 5 cells
  • FS4 cells a human hepatoma line
  • Hep G2 human hepatoma line
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et ai, PNAS USA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0.
  • CHO Chinese hamster ovary
  • DHFR-CHO cells Urlaub et ai, PNAS USA 77:4216 (1980)
  • myeloma cell lines such as NSO and Sp2/0.
  • mammalian ceil expression systems include CHO and HEK293-celi based expression systems.
  • Mammalian expression systems can utilize attached cell lines, for example, in T- fiasks, roller bottles, or cell factories, or suspension cultures, for example, in 1 L and 5L spinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50L and 100/200L WAVE bioreactors, among others known in the art.
  • a common bacterial host is £. coii.
  • the expression of proteins in prokaryotic cells such as E. coii is well established in the art. For a review, see for example Piuckthun, A. Bio/Technology. 9:545-551 (1991 ). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for recombinant production of polypeptides (see Ref, Curr. Opinion Biotech. 4:573-576, 1993; and Trill et ai., Curr. Opinion Biotech. 6:553-560, 1995).
  • protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series).
  • These and related embodiments may utilize the expression host strain BL21 (DE3), a X.DE3 lysogen of BL21 that supports T7 ⁇ mediated expression and is deficient in ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coii, such as Rosetta" (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling may also be improved using reagents such as Benzonase® nuclease and BugBuster® Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems.
  • Media of this type gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG.
  • Particular embodiments employ hexahistidine tags (such as His » Tag® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques.
  • clinical grade proteins can be isolated from £. coli inclusion bodies, with or without the use of affinity tags (see . , e.g., Shimp ef a/., Protein Expr Punt. 50:58-67, 2008).
  • certain embodiments may employ a cold-shock induced E. co!i high-yield production system, because over-expression of proteins in
  • Escherichia coii at low temperature improves their solubility and stability (see, e.g., Qing ef a/., Nature Biotechnology. 22:877-882, 2004).
  • a host celi strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, post- transiationai modifications such as acetylation, carboxylation, glycosyiation, phosphorylation, lipidation, and acylation.
  • Post-franslational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the protein of interest.
  • polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which, successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the celi type. Transient production, such as by transient transfection or infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include HEK293 and CHO-based systems.
  • Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from ceil culture.
  • Certain specific embodiments utilize serum free cell expression systems. Examples include HE 293 ceils and CHO cells that can grow on serum free medium (see, e.g., Rosser ef a/., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent number 6,210,922).
  • the proiein(s) produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art, Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) ⁇ e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography ⁇ e.g., Q, 8), size exclusion
  • chimeric ADi proteins Also included are methods of concentrating recombinantly produced proteins, e.g., chimeric ADi proteins.
  • methods of concentrating recombinantly produced proteins e.g., chimeric ADi proteins.
  • examples include iyophiiization, which is typically employed when the solution contains few soluble components other than the protein of interest.
  • Lyophilization is often performed after HPLC run, and can remove most or all volatile components from the mixture. Also included are ultrafiltration techniques, which typically employ one or more selective permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.
  • the chimeric ADi proteins have a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain therapeutic compositions, the chimeric ADI proteins have a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the chimeric ADi proteins have a purity of at least about 97% or 98% or 99%. in other embodiments, such as when being used as reference or research reagents, proteins can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.
  • the compositions described here are about substantially endotoxin free, including, for example, about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free.
  • the presence of endotoxins can be detected according to routine techniques in the art, as described herein, in specific embodiments, the chimeric ADi proteins are made from a eukaryotic ceil such as a mammalian or human ceil in substantially serum free media.
  • Another aspect of the invention provides a composition comprising one or more of the recombinant chimeric ADi described herein and a physiologically acceptable carrier.
  • the composition may further comprise an autophagy modulator
  • Autophagy modulators include, but are not limited to chloroquine, 3-methyiadenine, hydroxychloroquine, bafiiomycin A1 , 5-amino-4-imidazoie carboxamide riboside (AICAR), okadaic acid, N6-mercaptopurine riboside, vinblastine, wortmannin, rapamycin, everoiimus, metformin, perifosine, resveratrol, and tamoxifen.
  • the compositions comprising the recombinant chimeric ADI described herein may further comprise a chemotherapeutic agent, such as but not limited to docetaxei, carbopiatin,
  • cyclophosphamide gemcitabine, cisplatin, sorafenib, sunitinib or everoiimus, or
  • Yet another aspect of the present invention provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising one or more of the recombinant chimeric ADi described herein and a
  • the cancer can include, but is not limited to melanoma, pancreatic cancer, prostate cancer, small cell lung cancer,
  • mesothelioma lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer.
  • NSCLC non-small cell lung cancer
  • SEQ ID NO:1 is the amino acid sequence of wild type M. ho inis ADi.
  • SEQ ID NO:2 is the amino acid sequence of wild type M. arginini ADI.
  • SEQ ID NO:3 is the amino acid sequence of wild type M. arthritidis ADi.
  • SEQ ID NO:4 is the amino acid sequence of the DS1 recombinant chimeric
  • SEQ ID NO:5 is the amino acid sequence of the DS2 recombinant chimeric
  • SEQ ID NO:8 is the amino acid sequence of the DS3 recombinant chimeric ADI protein.
  • SEQ ID NO:7 is the amino acid sequence of the DS4 recombinant chimeric
  • SEQ ID NQ:8 is the amino acid sequence of a recombinant chimeric AD! derived from M. hominis Phoenix (see SEQ ID NO: 14; plus lysine substitutions) (catalytic domain) and M. arginini (a-helical domain).
  • SEQ ID NO:9 is the amino acid sequence of a recombinant chimeric ADi derived from M. hominis Phoenix (see SEQ ID NO: 14) (catalytic domain) and M. arthntidis (a-helicai domain).
  • SEQ ID NO: 10 is the amino acid sequence of the DS1 -1 lysine reduction mutant of the recombinant chimeric ADi protein (see Table E3).
  • SEQ ID NO:1 1 is the amino acid sequence of the DS1 -2 lysine reduction mutant of the recombinant chimeric ADI protein (see Table E3).
  • SEQ ID NO: 12 is the amino acid sequence of the DS1 -3 lysine reduction mutant of the recombinant chimeric ADi protein (see Table E3).
  • SEQ ID NO: 13 is the amino acid sequence of the DS1 -4 lysine reduction mutant of the recombinant chimeric ADi protein (see Table E3).
  • SEQ ID NO:14 is the amino acid sequence of the ADi Phoenix sequence. This ADI sequence is identical to M hominis ADI except for K1 12E and P210S substitutions.
  • SEQ ID NO: 15 is the amino acid sequence of M. aiiigatoris ADI.
  • SEQ ID NO:16 is the amino acid sequence of M. colombinum ADi.
  • SEQ ID NO:17 is the amino acid sequence of gailinarum ADi.
  • SEQ ID NO: 18 is the amino acid sequence of M, galea ADI.
  • SEQ ID NO: 19 is the amino acid sequence of M, iners ADL
  • SEQ ID NO:20 is the amino acid sequence of M, phocicerabra!e
  • ADL SEQ ID NO:21 is the amino acid sequence of M. phocidae ADI.
  • SEQ ID NOs:22-59 are the amino acid sequences of chimeric ADI proteins.
  • the present invention relates generally to chimeric ADi proteins, e.g., engineered to have reduced antigenicity as compared with corresponding wild type ADI molecules.
  • the present invention also relates to methods of treating cancer and other disorders with chimeric ADI, and in particular chimeric ADI-PEG 20.
  • Normal cells do not require arginine for growth, since they can synthesize arginine from citrulline in a two-step process catalyzed by ASS and ASL.
  • certain cancers do not express ASS.
  • Certain cancers do not express ASL, and other cancers may have diminished expression of, or may not express ASS and/or ASL. Therefore, these cancers are auxotrophic for arginine. This metabolic difference may be capitalized upon to develop a safe and effective therapy to treat these forms of cancer, ADI catalyzes the conversion of arginine to citrulline via the arginine dihydrolase pathway, and may thus be used to eliminate arginine.
  • Statistical significance it is meant that the result was unlikely to have occurred by chance.
  • Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance Ievel, then the null hypothesis is rejected, in simple cases, the significance level is defined at a p-value of 0.05 or less.
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, !ipofection).
  • Enzymatic reactions and purification techniques may be performed according to
  • Patient refers to an animal, in certain embodiments a mammal, and in a specific embodiment, a human.
  • Biocompatible refers to materials or compounds which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.
  • reference sequence refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.
  • PEG polyethylene glycol
  • ADI arginine deiminase
  • SS succinimidyl succinate
  • SSA succinimidyl succinimide
  • SPA succinimidyl propionate
  • NHS N-hydroxy-succinimide
  • ASS1 or ASS argininosuccinate synthetase
  • ASL argininosuccinate lyase.
  • the chimeric ADI enzymes as described herein are compared to the benchmark ADI-PEG 20 molecule derived from hominis.
  • ADI-PEG 20 refers to the ADI molecule known in the art and described for example in US6183738; US8635462; Ascierto PA, et ai. (2005) Pegyiated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and ⁇ studies.
  • this molecule is a pegylated (PEG 20,000) ADI enzyme derived from M. hominis, and has two substitutions (K1 12E; P210S) relative to the wild type M. hominis ADI enzyme.
  • polypeptide protein and peptide and “enzyme” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The terms do not exclude modifications such as myristoyiation, sulfation, glycosyiation, phosphorylation and addition or deletion of signal sequences.
  • polypeptide or protein or “enzyme” mean one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant ceils, or genetically-engineered or recombinant ceils, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
  • polypeptide and protein specifically encompass the chimeric ADI enzymes of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of the chimeric ADI enzymes.
  • the polypeptide is a "recombinant" polypeptide, produced by recombinant ceil that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.
  • isolated protein means that a subject protein (1 ) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovending interaction) with portions of a protein with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or noncovIER interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof.
  • the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • a chimeric ADI or a polynucleotide encoding a chimeric ADI may be derived, cloned or produced from any source, including, for example, microorganisms, recombinant biotechnology or any combination thereof.
  • arginine deiminase may be cloned from microorganisms of the genera Mycoplasma.
  • arginine deiminase is cloned from Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma phocicerebraie, Mycoplasma oraie, Mycoplasma galeae, Mycoplasma phocidae,
  • the ADI used in a chimeric ADI may comprise the amino acid sequence from Table A1 (any one of
  • MSVFDSKFKGIHVYSETGELESVLVHEPGRE I DYI TPARLDELLFSAILESH 5 DAR EHKQFVAELKANDINWELTDLVAETYDLASRAAKEEFIETFLEETVP VLTEANREAVRAFLLSKPTHEMVEFMMSGITKYELGVESE ELIVDPMPNLY FTRDPFASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLINTP YYDPSLK
  • MSVFDSKFNGTHVYSEIGELESVIA/HEPGREIDYITPARLDELLFSAILESHDAR 39 KEHKLFVSELKANDIN ELSDLVAETYTYI-IATQKEREAFIEKWLDEAEPALTKD LRAKVKSyVLSKEGTPVAMVRTMMAGVSKQELNVESETELWDPMPNLYFTRDPF ASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTPWYYDPSLKLSIEGGDVF
  • NNETLVVGVSERTDLDTI LLAKNIKANKEREFKRIVATNVPKWTNLMHLDTWLT MLDKDKfc'LYSPIA DVFKF DYDLV GGDEPOPKV GLPLEKLLESIINKKPlLl PIAGTSASNIDVERETHFDGTNYLAIAPGVVIGYSRNVKTNEALEAAGIKVLPFK.
  • the chimeric ADI comprises, consists, or consists essentially of an iilustrative chimeric sequence from Table A2 (SEQ ID NOs:4-13 or 22-59), or a variant or fragment thereof having ADI activity.
  • Certain embodiments include variants of the reference ADI polypeptide sequences described herein, whether described by name or by reference to a sequence identifier (e.g., Tables A1-A2).
  • a 'Variant" sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence disclosed herein by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions.
  • variants thus include fragments of a reference sequence described herein.
  • Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.
  • a biologically active variant will contain one or more conservative substitutions.
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since if is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittie, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doo!ittie, 1982).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1 ); giutamate (+3.0 ⁇ 1 ); serine (+0.3); asparagine (+0.2); g!utamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (-0.5); histidine ( ⁇ 0.5); cysteine (-1.0); methionine (-1 .3); valine (-1.5); leucine (-1.8); isoieucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; giutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoieucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophi!icity values include leucine, isoieucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1 ) aia, pro, giy, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, lie, leu, met, aia, phe; (4) iys, arg, his; and (5) phe, tyr, trp, his.
  • variant polypeptides may also, or alternatively, contain non-conservative changes.
  • variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.
  • a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620
  • a polypeptide sequence consists of about or no more than about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,
  • a polypeptide sequence is about 10-1000,
  • region of any reference polypeptide may be truncated by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400- 450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101 , 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide.
  • variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identify or sequence homology to a reference polypeptide sequence.
  • variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). in other embodiments, variant polypeptides differ from a reference sequence by at least 1 % but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.)
  • sequence similarity or sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, in a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (J. Moi. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Biossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Biossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:1 1-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a
  • “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLAST and X BLAST programs (version 2.0) of Altschul, et a/., (1990, J. Moi. Bioi, 215: 403-10).
  • Gapped BLAST can be utilized as described in Altschul et al., ⁇ Nucleic Acids Res. 25: 3389-3402, 1997).
  • the default parameters of the respective programs e.g., XBLAST and N BLAST.
  • polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool.
  • a local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared.
  • a modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs).
  • HSPs high-scoring segment pairs
  • the results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.
  • the raw score, S is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).
  • Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty.
  • the gap cost would be G+Ln.
  • the choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 1 1 , and a low value for L (1-2) e.g., 1 .
  • bit score S !
  • S is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches.
  • bit score and “similarity score” are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.
  • the E-Value or expected value, describes the likelihood that a sequence with a similar score will occur in the database by chance, it is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance.
  • the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related.
  • BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties.
  • BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 1 1 and a gap extension penalty of 1
  • sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 1 1 and a gap extension penalty of 1.
  • sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Acceirys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA.
  • GAP uses the algorithm of Needleman and Wunsch ( J Mo! Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 280, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
  • a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et a!., (Methods in Enzymo!. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D.
  • REM recursive ensemble mutagenesis
  • Native ADI may be found in microorganisms and is immunogenic and rapidly cleared from circulation in a patient. These problems may be overcome by engineering ADi to reduce its antigenicity, such as by engineering chimeric ADi molecules. In one
  • chimeric ADi are constructed by combining different domains (e.g. catalytic domain and a-heiicai domains) from different ADi proteins using standard molecular biological, or protein synthesis techniques.
  • the catalytic domain from M. arginini or M. arthritidis is combined with the a-heiical domain of M. hominis.
  • the catalytic domain of M. arginini is combined with the a-helical domain of M. arthritidis.
  • the catalytic domain of M. arthritidis is combined with the a-helicai domain of M. arginini.
  • catalytic and a-helical domains can be constructed from ADI proteins derived from other species, such as from Mycoplasma pneumoniae, Steptococcus pyogenes, Steptococcus pneumoniae, Borreiia burgdorferi, Borreiia afzeiii, Giardia intestinaiis, Clostridium perfringens, Bacillus iicheniformis, Enterococcus faecaiis, and Lactobacillus sake.
  • ADI proteins derived from other species such as from Mycoplasma pneumoniae, Steptococcus pyogenes, Steptococcus pneumoniae, Borreiia burgdorferi, Borreiia afzeiii, Giardia intestinaiis, Clostridium perfringens, Bacillus iicheniformis, Enterococcus faecaiis, and Lactobacillus sake.
  • Antigenicity problems may also be overcome by modifying chimeric ADI.
  • the present disclosure provides chimeric ADi modified by a modifying agent, including, but not limited to macromolecule polymers, proteins, peptides, polysaccharides, or other compounds. Arginine deiminase or chimeras thereof as described herein and the modifying agent may be linked by either covalent bonds or non-covalent interaction to form a stable conjugate or a stable composition to achieve a desired effect, in certain embodiments, the modified chimeric ADI retains the biological activity of an unmodified chimeric ADI and has a longer half-life in vivo and lower antigenicity than the unmodified, chimeric ADI.
  • the modified chimeric ADI retains at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 80%, 85%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the biological activity of unmodified chimeric ADi.
  • a modifying agent can be a polymer or a protein or a fragment thereof that is biocompatible and can increase the half-life of chimeric ADi in blood.
  • the modifying agent can be either chemically coupled to chimeric ADi or where applicable, linked to the chimeric ADI via fusion protein expression.
  • Macromo!ecuie polymers may include a non-peptide macromolecu!e polymer, which in certain embodiments, may have its own bioactivity.
  • Suitable polymers include, but are not limited to, polyenoi compounds, poiyether compounds, polyvinylpyrrolidone, poly amino acids, copolymer of divinyi ether and maleic anhydride, N-(2-hydroxypropy1) ⁇ methacrylamide, polysaccharide, polyoxyethylated po!yo!, heparin or its fragment, poly-aikyi- ethylene glycol and its derivatives, copolymers of poly-alkyl-ethylene glycol and its derivatives, polyvinyl ethyl ether), a,P-Po!y[(2-hydroxyethy!-DL-aspartamide].
  • polycarboxylates poly oxyethyiene-oxymethy!enes, poiyacryloyl morpholines, copolymer of amino compounds and oxyolefin, poly hyaluronic acid, polyoxiranes, copolymer of ethanedioic acid and maionic acid, poly (1 ,3-dioxoiane), ethylene and maleic hydrazide copolymer, poly sialic acid, cyclodextrin, etc. in certain embodiments, the polymer is polyethylene glycol.
  • polyenoi compounds as used herein include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxyi polyethylene glycol), polyvinyl alcohol, polyailyi alcohol, polybuteno! and the like, and their derivatives, such as lipids.
  • the poiyether compounds include, but are not limited to poly alkylene glycol (HO((CH2) x O) n H), polypropylene glycol, polyoxyrehylene (HO((CH 2 ) 2 0) n H), polyvinyl alcohol ((CH 2 CHOH) n ).
  • Poly amino acids include, but are not limited to, polymers of one type of amino acid or copolymers of two or more types of amino acids, for example, polyalanine or polylysine, or block co-polymers thereof.
  • Polysaccharides include but are not limited to, glucosan and its derivatives, for example dextran sulfate, cellulose and its derivatives (including methyl cellulose and carboxymethyi cellulose), starch and its derivatives, polysucrose, etc.
  • chimeric ADI is modified by coupling with proteins or peptides, wherein one or more proteins or peptides are directly or indirectly linked to chimeric ADi.
  • the proteins can either be naturally existing proteins or their fragments, including but not limited to naturally existing human serum proteins or their fragments, such as thyroxine-binding protein, transthyretin, a1-acid glycoprotein, transferrin, fibrinogen, immunoglobulin, ig Fc regions, albumin, and fragments thereof.
  • fragment is meant any portion of a protein that is smaller than the whole protein but which retains the desired function of the protein.
  • Engineered chimeric ADl may be directly or indirectly linked to a protein via a covalent bond.
  • Direct linking means that one amino acid of chimeric ADl is directly linked to one amino acid of the modifying protein, via a peptide bond or a disulfide bridge
  • indirect linking refers to the linkages between a chimeric AD! and a modifying protein, via originally existing chemical groups there between or specific chemical groups added through biological or chemical means, or the combination of the above-mentioned linkages.
  • chimeric ADl is modified by covalent attachment with PEG.
  • Chimeric ADl covalentiy modified with PEG may be hereinafter referred to as chimeric "ADi-PEG.”
  • chimeric ADi-PEG When compared to unmodified chimeric ADl, chimeric ADi-PEG retains most of its enzymatic activity, is far less antigenic, has a greatly extended circulating half-life, and is much more efficacious in the treatment of tumors.
  • Polyethylene glycol or “PEG” refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH 2 CH 2 )nOH, wherein n is at least 4.
  • Polyethylene glycol or “PEG” is used in combination with a numeric suffix to indicate the approximate weight average molecular weight thereof.
  • PEGS, 000 refers to PEG having a total weight average molecular weight of about 5,000
  • PEG12,000 refers to PEG having a total weight average molecular weight of about 12,000
  • PEG20,000 refers to PEG having a total weight average molecular weight of about 20,000.
  • the PEG has a total weight average molecular weight of about 1 ,000 to about 50,000; in one embodiment from about 3,000 to about 40,000, and in another embodiment from about 5,000 to about 30,000; in certain embodiments from about 8,000 to about 30,000; in other embodiments from about 1 1 ,000 to about 30,000; in additional embodiments, from about 12,000 to about 28,000; in still other embodiments, from about 16,000 to about 24,000; and in other embodiments, about 18,000 to about 22,000; in another embodiment, from 19,000 to about 21 ,000, and in one embodiment, the PEG has a total weight average molecular weight of about 20,000.
  • PEG with a molecular weight of 30,000 or more is difficult to dissolve, and yields of the formulated product may be reduced.
  • the PEG may be a branched or straight chain.
  • increasing the molecular weight of the PEG decreases the antigenicity of the ADl or chimeric ADl.
  • the PEG having a molecular weight described in this embodiment may be used in conjunction with chimeric ADl, and, optionally, a biocompatible linker, to treat cancer, including, for example, acute myeloid leukemia, such as relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, stomach cancer and esophageal cancer.
  • acute myeloid leukemia such as relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head
  • the PEG has a total weight average molecular weight of about 1 ,000 to about 50,000; in certain embodiments about 3,000 to about 30,000; in other embodiments from about 3,000 to about 20,000; in one embodiment from about 4,000 to about 12,000; in still other embodiments from about 4,000 to about 10,000; in additional embodiments from about 4,000 to about 8,000; still further embodiments from about 4,000 to about 6,000; and about 5,000 in another embodiment.
  • the PEG may be a branched or straight chain, and in certain embodiments is a straight chain.
  • the PEG having a molecular weight described in this embodiment may be used in conjunction with chimeric AD!, and optionally, a biocompatible linker, to treat graft versus host disease (GVHD) or cancer.
  • GVHD graft versus host disease
  • chimeric ADI-PEG is the illustrative modified chimeric AD! described herein, as would be recognized by the skilled person chimeric AD! may be modified with other polymers or appropriate molecules for the desired effect, in particular reducing antigenicity and increasing serum half-life.
  • Chimeric ADI may be covalently bonded to a modifying agent, such as PEG, with or without a linker, although a preferred embodiment utilizes a linker.
  • a modifying agent such as PEG
  • linker used to covalently attach chimeric ADI to a modifying agent e.g.
  • PEG may be any biocompatible linker.
  • biocompatible indicates that the compound or group is non-toxic and may be utilized in vitro or in vivo without causing injury, sickness, disease, or death.
  • a modifying agent, such as PEG can be bonded to the linker, for example, via an ether bond, a thiol bond, or an amide bond.
  • the linker group includes, for example, a succinyi group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, and combinations thereof, in one embodiment, the source of the biocompatible linker is succinimidyl succinate (SS).
  • SS succinimidyl succinate
  • linker may include an oxycarbonylimidazole group (including, for example, carbonylimidazole (GDI)), a nitro phenyl group (including, for example, nitrophenyi carbonate (NCP) or trichiorophenyl carbonate (TCP)), a trysylate group, an aldehyde group, an isocyanafe group, a vinylsuifone group, or a primary amine, in another embodiment, the linker is derived from SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS are used, and in other embodiments, SS or SPA are used.
  • potential linkers can be formed from methoxy-PEG succinimidyl
  • chimeric ADI may be coupled directly to a modifying agent, such as PEG (i.e., without a linker) through an amino group, a sulfhydryi group, a hydroxyl group or a carboxyl group.
  • a modifying agent such as PEG (i.e., without a linker) through an amino group, a sulfhydryi group, a hydroxyl group or a carboxyl group.
  • Chimeric ADi may be covalently bonded to PEG, via a biocompatible linker, using methods known in the art, as described, for example, by Park et ai, Anticancer Res., 1 :373-376 (1981 ); and Zapiipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992), the disclosures of which are hereby incorporated by reference herein in their entirety.
  • PEG polystyrene-maleic anhydride
  • a primary amine of chimeric ADI Selection of the attachment site of PEG, or other modifying agent, on the chimeric ADi is determined by the role of each of the sites within the active domain of the protein, as would be known to the skilled artisan.
  • PEG may be attached to the primary amines of chimeric ADi without substantial loss of enzymatic activity. For example, ADi cloned from Mycoplasma arginini, Mycoplasma arthritidis and Mycoplasma hominis has a number of lysine residues that may be modified by this procedure.
  • one or more or all of the lysines are possible points at which ADI and chimeric forms of ADi as described herein can be attached to PEG via a biocompatible linker, such as SS, SPA, SCM, SSA and/or NHS.
  • PEG may also be attached to other sites on AD! and chimeric forms of ADI as described herein, as would be apparent to one skilled in the art in view of the present disclosure.
  • PEG molecules may be covalently bonded to chimeric
  • chimeric ADI is modified with one PEG molecule. In other embodiments, chimeric ADI is modified with more than one PEG molecule. In one embodiment, chimeric ADi is modified with about 1 to about 10 PEG molecules, in one embodiment from about 2 to about 8 PEG molecules and in another embodiment, from about 9 to about 12 PEG molecules, in another embodiment, the chimeric ADI is modified with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 PEG molecules, in one specific embodiment, chimeric AD! is modified with 4.5 - 5.5 PEG molecules per ADi. in another embodiment, chimeric ADi is modified with 5 ⁇ 1.5 PEG molecules.
  • about 15% to about 70% of the primary amino groups in chimeric ADi are modified with PEG, in one embodiment about 20% to about 85%, about 25% to about 60%, or in certain embodiments about 30% to about 55%, or 45% to about 50%, and in other embodiments about 20% or 30% or 40% or 50% of the primary amino groups in arginine deiminase are modified with PEG.
  • the range of primary amino groups depends upon how many lysines are successfully removed. In certain embodiments, all of the lysines may be removed and the N- terminus of the molecule is PEGyiated. When PEG is covalentiy bonded to the end terminus of chimeric ADI, it may be desirable to have only 1 PEG molecule utilized. Increasing the number of PEG units on chimeric ADI increases the circulating half-life of the enzyme.
  • a common feature of biocompatible linker is that they attach to a primary amine of arginine deiminase via a succinyl group.
  • SS-PEG Once coupled with chimeric ADI, SS-PEG has an ester linkage next to the PEG, which may render this site sensitive to serum esterase, which may release PEG from chimeric ADI in the body.
  • SPA- PEG and PEG2-NHS do not have an ester linkage, so they are not sensitive to serum esterase.
  • a biocompatible linker is used in the present invention.
  • PEG which is attached to the protein may be either a straight chain, as with SS- PEG, SPA-PEG and SC-PEG, or a branched chain of PEG may be used, as with PEG2- NHS.
  • the chimeric ADI of the present disclosure may be modified as described in US Patent No. 6,835,462.
  • modifications of one or more of the naturally occurring amino acid residues of ADI and chimeric molecules of ADI in particular derived from Mycoplasma hominis, M, arthritidis and M. arginini, can provide for an enzyme that is more easily renatured and formulated thereby improving existing techniques for the manufacture of chimeric ADI and therapeutic compositions comprising the same.
  • the chimeric ADI of the present disclosure is modified to remove one or more lysine residues (e.g., the lysine can be substituted with another amino acid or analogues thereof, or a non-natural amino acid).
  • the chimeric ADI is modified to be free of the lysine at position 1 12, 374, 405 or 408 of SEQ ID NO:1 (M. hominis ADI), or a combination of one or more of these positions, in a further embodiment, the chimeric ADI is modified to be free of one or more lysines, for example, 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or more lysine residues, should they be present, can be substituted with another amino acid or analogues thereof, or a nonnatural amino acid.
  • a chimeric ADI has 5 lysines substituted, for example, at position 7, 88, 137, 209, and 380 of SEQ ID NO: 4.
  • a chimeric ADl has 10 lysines substituted, for example, at positions 7, 9, 59, 88, 1 15, 1 16, 137, 178, 209, and 380 of SEQ ID NO: 4,
  • a chimeric ADl has 15 lysines substituted, for example, at positions 7, 9, 59, 66, 88, 91 , 93, 1 15, 1 16, 137, 141 , 178, 209, 279, and at position 380 of SEQ ID NO: 4.
  • a chimeric ADl comprises 21 lysines substituted, for example, at positions 7, 9, 56, 59, 66, 88, 91 , 93, 96, 1 15, 1 16, 137, 141 , 178, 209, 254, 279, 325, 326, 380, and 406 of SEQ ID NO: 4,
  • Illustrative chimeric ADl molecules having lysine substitutions are set forth in SEQ ID NOs: 10-13.
  • pegylation sites associated with ADl located at or adjacent to the catalytic region of the enzyme are modified.
  • the phrase "pegylation site” may be defined as any site or position of AD! or a chimeric ADl that may be covendingly modified with polyethylene glycol
  • a "pegylation site” can be considered located at or adjacent to the catalytic region of the enzyme where pegylation of the site results in a significant reduction in catalytic activity of the enzyme. The pegylation of such sites has traditionally resulted in the inactivation of the enzyme.
  • ADl from Mycoplasma hominls has a lysine at the 1 12 position which can be considered to be at or adjacent the catalytic region of the enzyme. The attachment of PEG to this lysine at the 1 12 position can inactivate the enzyme, in addition, ADl from
  • Mycoplasma homlnis has a cysteine at the 397 position which can be considered to be at or adjacent the catalytic region of the enzyme.
  • the amino acid substitutions for cysteine at the 397 position can inactivate the enzyme.
  • substituting alanine, histidine, arginine, serine, lysine or tyrosine for cysteine at the 397 position can result in a loss of ail detectable enzyme activity.
  • ADl from Mycoplasma homlnis also has three lysines located near this conserved cysteine, in particular Lys374, Lys405 and Lys408. The attachment of PEG to Lys374, Lys405, Lys408 or combinations thereof can inactivate the enzyme.
  • ADl derived from other organisms may also have pegylation sites corresponding to 1 12 position of ADl from Mycoplasma hominls.
  • ADl from Steptococcus pyrogenes has lysine at the 104 position
  • ADl from Steptococcus pyrogenes has lysine at the 104 position
  • Mycoplasma pneumoniae has lysine at the 106 position
  • ADl from Giardia intestinaiis has lysine at the 1 14 position
  • AD! from some organisms may have lysines corresponding to the same general location as the 1 12 position of ADl from Mycoplasma homlnis, The location of lysine in ADl from such organisms are known to the skilled person and are described in US Patent No. 6,635,462.
  • the present invention provides for certain amino acid substitutions in the polypeptide chain of AD!. These amino acid substitutions provide for modified ADl that loses less activity when modified by a modifying agent, e.g., upon pegy!ation. By eliminating pegyiation sites, or other known modification sites, at or adjacent to the catalytic region of enzyme, optima! modification, e.g., pegyiation, can be achieved without the loss of activity.
  • arginine deiminase may prevent or interfere with the proper and rapid renaturation when produced via recombinant technology, in particular, these structural characteristics hinder or prevent the enzyme from assuming an active conformation during recombinant production.
  • active conformation may be defined as a three-dimensional structure that allows for enzymatic activity by unmodified or modified arginine deiminase or chimeric arginine deiminase. The active conformation may, in particular, be necessary for catalyzing the conversion of arginine into citru!iine.
  • structural characteristic may be defined as any trait, quality or property of the polypeptide chain resulting from a particular amino acid or combination of amino acids.
  • arginine deiminase may contain an amino acid that results in a bend or kink in the normal peptide chain and thus hinders the enzyme from assuming an active conformation during renaturation of the enzyme.
  • arginine deiminase from Mycoplasma hominis has a proline at the 210 position that may result in a bend or kink in the peptide chain, making it more difficult to renature the enzyme during recombinant production, it is to be understood that arginine deiminase derived from other organisms may also have sites corresponding to the 210 position of arginine deiminase from Mycoplasma hominis.
  • the present invention thus again provides for certain amino acid substitutions in the polypeptide chain of wild type arginine deiminases and chimeric arginine deiminases derived therefrom.
  • Such amino acid substitutions can eliminate the problematic structurai characteristics in the peptide chain of arginine deiminase.
  • Such amino acid substitutions provide for improved renaturation of the modified arginine deiminase.
  • These amino acid substitutions make possible rapid renaturing of modified chimeric arginine deiminases using reduced amounts of buffer.
  • These amino acid substitutions may also provide for increased yields of renatured modified chimeric arginine deiminase.
  • the modified chimeric arginine deiminase has an amino acid substitution at P210 or the equivalent residue.
  • Mycoplasma hominis has the amino acid proline located at the 210 position. While not limiting the present invention, it is presently believed that the presence of the amino acid proline at position 210 results in a bend or kink in the normal polypeptide chain that increases the difficulty of renaturing (i.e., refolding) arginine deiminase. Substitutions for proline at position 210 make possible the rapid renaturation of modified arginine deiminase and chimeras derived therefrom using reduced amounts of buffer. Substitutions for proline at position 210 may also provide for increased yields of renatured modified chimeric arginine deiminase. In one embodiment, the proline at position 210 is substituted with serine. It is to be understood that in accordance with this aspect of the invention, other substitutions at position 210 may be made. Examples of other substitutions include Pro210 to Thr210.
  • Pro210 to Arg21 G, Pro210 to Asn21 G, Pro210 to Gln210 or Pro210 to Met210 By eliminating those structural characteristics associated with the amino acid of position 210 of the wild- type arginine deiminase and chimeras derived therefrom, proper refolding of the enzyme can be achieved.
  • the methods of the present invention can involve either in vitro or in vivo applications, in the case of in vitro applications, including ceil culture applications, the compounds described herein can be added to the cells in cultures and then incubated.
  • the compounds of the present invention may also be used to facilitate the production of monoclonal and/or poiycional antibodies, using antibody production techniques well known in the art.
  • the monocionai and/or poiycional antibodies can then be used in a wide variety of diagnostic applications, as would be apparent to one skilled in the art.
  • the in vivo means of administration of the compounds of the present invention will vary depending upon the intended application.
  • Administration of the chimeric ADI compositions described herein, in pure form or in an appropriate pharmaceutical composition can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • the pharmaceutical compositions can be prepared by combining chimeric ADI, e.g., chimeric ADI-PEG, chimeric ADI-PEG 20, with an appropriate
  • physiologically acceptable carrier diluent or excipient
  • diluent or excipient may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
  • chimeric ADI-PEG e.g., chimeric ADI-PEG 20
  • parenteraliy intravenously, intraiymphatical!y, intratumorly, intramuscularly, interstitia!iy, intra-arterially, subcutaneously, intraoculariy, intrasynoviai, transepithelial, and
  • the chimeric ADi compositions herein increase median survival time of patients by a statistically significant amount.
  • the chimeric ADI treatments described herein increase median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer.
  • chimeric ADi treatments increase median survival time of a patient by 1 year, 2 years, 3 years, or longer, in one embodiment, the chimeric ADi treatments described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer, in certain embodiments, the chimeric ADI treatments described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.
  • the amount administered is sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 50% decrease in tumor mass, or by altered ⁇ e.g., decreased with statistical significance) scan dimensions.
  • the amount administered is sufficient to result in stable disease.
  • the amount administered is sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.
  • the amount administered is sufficient to inhibit NO synthesis, inhibit angiogenesis, and or is sufficient to induce apoptosis in tumor cells or any combination thereof.
  • NO synthesis, angiogenesis and apoptosis may be measured using methods known in the art, see, e.g., Current Protocols in immunoiogy or Current Protocois in Molecular Biology, John Wiley & Sons, New York, N.Y.(2009 and updates thereto); Ausubel et a/., Short Protocols in Molecular Biology, 3" 1 ed., Wiley & Sons, 1995; and other like references.
  • the amount administered inhibits NO synthesis and inhibits the growth of melanoma and synergizes with other chemotherapies as described herein, such as cispiatin.
  • cispiatin chemotherapies as described herein, such as cispiatin.
  • one embodiment of the present disclosure provides a method of treating melanoma by administering chimeric ADI-PEG 20 in combination with cispiatin, wherein the treatment depletes endogenous nitric oxide (NO).
  • the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated.
  • a pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects.
  • the composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.
  • the chimeric ADI compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy,
  • compositions may also be administered in combination with antibiotics.
  • Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavaiiable upon administration of the composition to a patient.
  • compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described chimeric ADI composition in aerosol form may hold a plurality of dosage units.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • the composition to be administered will, in any event, contain a therapeutically effective amount of a chimeric AD!-PEG of the present disclosure, such as chimeric ADI-PEG 20, for treatment of a disease or condition of interest in accordance with teachings herein.
  • a pharmaceutical composition may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, anoral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition is generally either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystailine cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as aiginic acid, sodium alginate, Primogei, corn starch and the like; lubricants such as magnesium stearate or Sterotex; giidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol or oil.
  • the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer, in a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, in certain embodiments, physiological saline, Ringers solution, isotonic sodium chloride, fixed oils such as synthetic mono or digiycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethy!enediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvants.
  • a liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of chimeric ADI as herein disclosed, such as chimeric ADI-PEG 20, such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 % of chimeric ADI in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of chimeric ADI-PEG. in certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of chimeric ADI-PEG prior to dilution.
  • the pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emuisifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration, if intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • the pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the pharmaceutical composition in solid or liquid form may include an agent that binds to chimeric AD!-PEG and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
  • the pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers. and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.
  • compositions may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining a composition that comprises chimeric ADI-PEG as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-cova!entiy interact with the chimeric ADI-PEG composition so as to facilitate dissolution or homogeneous suspension of the chimeric ADI-PEG in the aqueous delivery system.
  • the com positions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound (e.g., chimeric ADI-PEG) employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • the specific compound e.g., chimeric ADI-PEG
  • a therapeutically effective amount of one of the compounds of the present invention is an amount that is effective to inhibit tumor growth.
  • treatment is initiated with small dosages which can be increased by small increments until the optimum effect under the circumstances is achieved.
  • a therapeutic dosage of compounds of the present invention may be from about 1 to about 200 mg/kg twice a week to about once every two weeks.
  • the dosage may be about 1 mg/kg once a week as a 2 ml intravenous injection to about 20 mg/kg once every 3 days.
  • the dose may be from about 50 !U/m 2 to about 8,000 IU/m 2 , administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks, in certain embodiments, the dose may be about 50 IU/m 2 , 60 !U/m 2 , 70 IU/m 2 , 80 !U/m 2 , 90 iU/m 2 , 100 IU/m 2 , 1 10 iU/rn 2 , 120 IU/m 2 , 130 IU/m 2 , 140 IU/m 2 , 150 IU/m 2 , 160 IU/m 2 , 170 IU/m 2 , 180 IU/m 2 , 190 IU/m 2 , 200 IU/m 2 , 210 IU/m 2 , 220 IU/m 2 , 230 IU/rn 2 , 240 IU/m 2 , 250 IU/rn 2 , 260 IU/m 2
  • I U/m 1 administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks.
  • the dose may be about 1 rng/m 2 , 2 mg/m 2 . 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 .
  • the dose may be modified as desired by the skilled clinician.
  • the dose may be modified as desired by the skilled clinician.
  • the optimum dosage with chimeric ADi-SS-PEG5,000 may be about twice a week, while the optimum dosage with chimeric ADI-SS ⁇ PEG20,000 may be from about once a week to about once every two weeks. In certain embodiments, the optimum dosage with chimeric ADI-SS-PEG20,000 may be about twice a week.
  • Chimeric ADI-PEG may be mixed with a phosphate buffered saline solution, or any other appropriate solution known to those skilled in the art, prior to injection.
  • a liquid composition comprising chimeric ADI-PEG comprises about 10 to about 12 mg of chimeric ADI, about 20 to about 40 mg of polyethylene glycol, 1.27 mg +5% monobasic sodium phosphate, USP; about 3 mg +5% dibasic sodium phosphate, USP; 7.6 mg +5% sodium chloride, USP; at a pH of about 6.6 to about 7; in an appropriate amount of water for injection (e.g., about 1 ml or about 2 ml).
  • a liquid composition comprising a chimeric ADI-PEG comprises histidine - HCI
  • the composition buffer is from about 0.0035M Histidine-HCi to about 0.35M Histidine-HCI.
  • the composition is formulated in a buffer comprising 0.035 M Histidine-HCI at pH 6.8 with 0.13 M sodium chloride, in another embodiment, the composition is formulated in a buffer comprising 0.02M sodium phosphate buffer at pH 6.8 with 0.13 M sodium chloride.
  • a composition comprising chimeric ADI or chimeric ADI- PEG has a pH of about 5 to about 9, about 6 to about 8, or about 6.5 to about 7.5. in some embodiments, the composition comprising chimeric ADI has a pH of about 6.8 ⁇ 1.0.
  • free PEG in a composition comprising chimeric ADI-PEG is between 1-10%, and in a further embodiment, is less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1 % of the total PEG.
  • the unmodified chimeric AD! in a composition comprising chimeric ADI-PEG is less than about 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less than 0.1 %.
  • compositions comprising chimeric ADI-PEG have total impurities less than or equal to about 4%, 3%, 2%, 1.5%, 1 % or 0.5%.
  • the endotoxin limit meets the requirements stated in USP, i.e., ⁇ 50 EU/mL,
  • the free sulfhydryi in a composition comprising chimeric ADI or chimeric ADI-PEG is greater than about 90%. In some embodiments, the free sulfhydryi in a composition comprising chimeric ADI or chimeric ADI-PEG is about 91 %, about 92%, about 93%, about 94% or about 95%, about 96% about 97%, about 98% about 99% or more.
  • the chimeric AD! or chimeric ADI-PEG in a composition has a Km of from about 0.5 ⁇ to about 15 ⁇ , and in a further embodiment, is from about 1 ⁇ to about 12 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1.5 ⁇ to about 9 ⁇ , about 1 .5 ⁇ to about 8 ⁇ or about 1.5 ⁇ to about 7 ⁇ .
  • the chimeric ADi or chimeric ADI-PEG in a composition has a Km of about 1.5 ⁇ to about 6.5 ⁇ .
  • the chimeric ADi or chimeric ADI-PEG in a composition has a Km of about 1.5 ⁇ , about 2 ⁇ , about 2.5 ⁇ , about 3 ⁇ , about 3.5 ⁇ , about 4 ⁇ , about 4.5 ⁇ , about 5 ⁇ , about 5.5 ⁇ , about 8 ⁇ , about 6.5 ⁇ , or about 7 ⁇ .
  • the chimeric ADI or chimeric AD!-PEG in a composition has a reduced Km compared to a wild- type AD! or wiid-type ADI-PEG in the composition.
  • the chimeric ADi or chimeric ADI-PEG in a composition has a Kcat of from about 0.5 sec "1 to about 15 sec "1 , and in a further embodiment, is from about 1 sec "1 to about 12 sec “1 , about 1 sec “1 to about 10 sec “1 , about 1.5 sec “1 to about 9 sec “1 , about 2 sec “1 to about 8 sec “1 or about 2.5 sec “1 to about 7 sec “1 , in certain
  • the chimeric ADI or chimeric ADI-PEG in a composition has a Kcat of about 2.5 sec "1 to about 7.5 sec "1
  • the chimeric ADi or chimeric ADI-PEG in a composition has a Kcat of about 2.5 sec "1 , about 3 sec “1 , about 3.5 sec “1 , about 4 sec “1 , about 4.5 sec “1 , about 5 sec “1 , about 5.5 sec "1 , about 6 sec “1 , about 6.5 sec “1 , about 7 sec “1 , about 7.5 sec “1 or about 8 sec “1
  • the chimeric ADI or chimeric ADI-PEG in a composition has a higher Kcat than a wild-type ADI or wild-type ADI-PEG in the composition.
  • the chimeric AD! or chimeric ADI-PEG in a composition has a conductivity (also referred to in the art as specific conductance) of about 5 mS/cm to about 20 mS/cm, and in further embodiments, from about 5 mS/cm to about 15 mS/cm, about 7 mS/cm to about 15 mS/cm, about 9 mS/cm to about 15 mS/cm or about 10 mS/cm to about 15 mS/cm.
  • a conductivity also referred to in the art as specific conductance
  • the chimeric ADI or chimeric ADI-PEG in a composition has a conductivity of about 9 mS/cm, about 10 mS/cm, about 1 1 mS/cm, about 12 mS/cm or about 13 mS/cm, about 14 mS/cm or about 15 mS/cm.
  • the chimeric ADi or chimeric ADI-PEG in a composition has a conductivity of about 13 mS/cm ⁇ 1.0 mS/cm.
  • the chimeric ADi or chimeric ADI-PEG in a composition has an osmolality of about 50 mOsm/kg to about 500 mOsm/kg, about 100 mOsm/kg to about 400 mOsm/kg, about 150 mOsm/kg to about 350 mOsm/kg, about 200 mOsm/kg to about 350 mOsm/kg or about 250 mOsm/kg to about 350 mOsm/kg.
  • the chimeric AD! or chimeric ADI-PEG in a composition has an osmolality of about 300 ⁇ 30 mOsm/kg,
  • the protein concentration is about 1 1.0 ⁇ 1 .0 mg/mL in certain embodiments, the protein concentration is between about 8 and about 15 mg/mL, In another embodiment, the protein concentration is about 8, 9, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 13, 13.5, 14, or 15 mg/mL.
  • the specific enzyme activity is between 5.0 and 150 !U/mg, where 1 IU is defined as the amount of enzyme that converts one moi of arginine into one mol of citrulline and 1 ⁇ ! of ammonia in one minute at 37° C and the activity is 100 ⁇ 20 iU/mg.
  • the specific enzyme activity is about 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.0, 9.5, 10, 10.5, 1 1 , 1 1 .5, 12, 12,5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, or about 150 ⁇ 2.0 IU/mg. In one particular embodiment, the specific enzyme activity is 100 ⁇ 10.0 IU/mg.
  • compositions comprising chimeric ADI-PEG of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents.
  • Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising chimeric ADI-PEG (e.g., chimeric ADI-PEG 20) of the invention and each active agent in its own separate pharmaceutical dosage formulation.
  • chimeric ADI-PEG as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
  • chimeric ADI-PEG as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations.
  • compositions comprising chimeric ADi-PEG and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
  • chimeric ADI compositions of this disclosure in combination with one or more other therapeutic agents
  • therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer or GVHD.
  • exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-infiammatories, chemotherapeutics, radiotherapeutics, autophagy modulators, or other active and ancillary agents.
  • the chimeric AD! compositions disclosed herein may be administered in conjunction with any number of chemotherapeutic agents.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); aikyi sulfonates such as busuifan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
  • methylamelamines including altretamine, triethyleneme!amine, trietylenephosphoramide, triethyienethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
  • bestrabucil bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
  • mitoguazone mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyliinic acid; 2-ethyihydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran;
  • vindesine dacarbazine; mannomustine; mitobronitoi; mito!actol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g.
  • paciitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ.) and docetaxei (TAXOTERE®., Rhne- Pouienc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carbop!atin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
  • navelbine novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; dif!uoromethyiomithine (DMFO); retinoic acid derivatives such as TargretinTM (bexarotene), PanretinTM (aiitretinoin) ; ONTAKTM (deni!eukin diftitox) ; esperamicins; capecitabine; and pharmaceuticaiiy acceptable salts, acids or derivatives of any of the above.
  • DMFO dif!uoromethyiomithine
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoies, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as f!utamide, ni!utamide, bica!utamide, ieuproiide, and goserelin.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoies, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as f!utamide, ni!utamide
  • chemotherapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib. bevacizumab, cetuximab, crizotinib, dasatinib, eriotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxoiitinib, trastuzumab, vandetanib, vemurafenib, and sunitinib; siroiimus (rapamycin), everolimus and other mTOR inhibitors.
  • Pharmaceutically acceptable salts, acids or derivatives of any of the above are also contemplated for use herein.
  • the chimeric ADI compositions disclosed herein may be administered in conjunction with any number of autophagy inhibitors, in some preferred embodiments, the autophagy inhibitor is selected from the group consisting of: chloroquine, 3-methyiadenine, hydroxychloroquine (Plaquenil.TM.), bafilomycin A1 , 5-amino-4-imidazoie carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1 , analogues of cAMP, and drugs which elevate cAMP levels, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine, in addition, antisense or siRNA that modulates expression of proteins essential for autophagy, such as for example ATG5, may also be used.
  • the autophagy inhibitor is selected from the group consisting of: chloroquine, 3-methyiadenine,
  • the combination of chimeric ADI-PEG with one or more therapeutic agents acts additively or synergisticaily.
  • synergizing agents include a therapeutic agent (e.g., chemotherapeutic agent, autophagy inhibitor, mTOR inhibitor, or any other therapeutic agent used for the treatment of cancer, GVHD, or inflammatory bowel disease as described herein) that is capable of acting synergisticaily with chimeric ADI-PEG as provided herein, where such synergy manifests as a detectable effect that is greater (i.e., in a statistically significant manner relative to an appropriate control condition) in magnitude than the effect that can be detected when the chemotherapeutic agent is present but the ADi-PEG composition is absent, and/or when the ADI-PEG is present but the chemotherapeutic agent is absent.
  • the com positions comprising chimeric ADi , and optionally other therapeutic agents, as described herein may be used in therapeutic methods for treating of cancer and methods for preventing metastasis of a cancer.
  • the present invention provides for methods for treating, ameliorating the symptoms of, or inhibiting the progression of or prevention of a variety of different cancers, in another embodiment, the present disclosure provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of GVHD.
  • the present disclosure provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer or GVHD in a patient comprising administering to the patient a therapeutically effective amount of chimeric ADi composition as described herein, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer or GVH D.
  • the chimeric ADI compositions described herein may be administered to an individual afflicted with inflammatory bowel disease (e.g., Crohn's disease; ulcerative colitis), GVHD or a cancer, including, but not limited to leukemia (e.g.
  • sarcomas including, but not limited to, metastatic sarcomas, uterine leiomyosarcoma
  • pancreatic cancer prostate cancer (such as, but not limited to, hormone refractory prostate cancer), mesothelioma, lymphatic leukemia, chronic myelogenous leukemia, lymphoma, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including, but not limited to, gastric adenocarcinoma), glioma, glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney cancer (including but not limited to renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers (including, but not limited to, squamous cell carcinoma of the head and neck
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of myeloid leukemia, such as, but not limited to, acute myeloid leukemia (A L), by administering a therapeutically effective amount of a chimeric ADI-PEG 20.
  • the myeloid leukemia such as AML
  • the present disclosure provides a method of treating AML comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks, in certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of AML is between about 50 !
  • the dose of chimeric ADI-PEG 20 administered for the treatment of AML is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 , 60 mg/m 2 , 65 mg/m 2 , 70 mg/m 2 , 75 mg/m 2 , or about 80 mg/m 2 .
  • the present disclosure provides a method of treating AML, wherein the dose of chimeric ADI is doubled and may be increased to 640 !U/m2 per week or more.
  • the chimeric AD! for the treatment of AML is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating AML by administering a composition comprising chimeric ADI-PEG 20 wherein the composition comprises a chimeric ADI modified with 5 ⁇ 1.5 PEG molecules, and in one embodiment, 5 ⁇ 1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., unmodified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of sarcomas, including but not limited to metastatic sarcomas, by administering a therapeutically effective amount of a chimeric ADI-PEG 20.
  • the sarcoma is deficient in ASS, ASL, or both.
  • the present disclosure provides a method of treating a sarcoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks.
  • the dose of chimeric ADI-PEG 20 administered for the treatment of sarcomas is between about 50 IU/m 2 and about 8,000 IU/m 2 , and in other embodiments is about 50 IU/m2, about 100 IU/m 2 , 150 !U/m 2 , 200 IU/m 2 , 250 IU/m 2 , 300 IU/m 2 , 350 IU/m 2 , 400 IU/m 2 , 450 IU/m 2 , 500 IU/m 2 , 550 IU/m 2 , 600 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 IU/m 2 , 800 IU/m 2 , about 900 IU/m 2 , about 1 ,000 IU/m 2 , 1 ,500 IU/m 2 about 2,000 IU/m 2 , about 2,500 IU/m 2 , about 3,000 IU/m 2 , 3,500 IU/m 2
  • the dose of chimeric ADI-PEG 20 administered for the treatment of sarcomas is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 , 60 mg/m 2 .
  • the present disclosure provides a method of treating sarcoma, wherein the dose of chimeric ADi is doubled and may be increased to 640 IU/m2 per week or more.
  • the chimeric ADi for the treatment of AML is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating a sarcoma, including a metastatic sarcoma, by administering a composition comprising chimeric ADI-PEG 20 wherein the composition comprises an chimeric ADi modified with 5 ⁇ 1.5 PEG molecules, and in one embodiment, 5 ⁇ 1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADi (i.e., unmodified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of pancreatic cancer by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with an autophagy inhibitor, such as but not limited to chloroquine, 3- methyiadenine, hydroxychloroquine, bafilomycin A1 , 5-amino-4-imidazoie carboxamide riboside (AICAR), okadaic acid, N6-mercaptopurine riboside, wortmannin, and vinblastine, in certain embodiments, the pancreatic cancer is deficient in ASS, ASL or both, in a further embodiment, the present disclosure provides a method of treating pancreatic cancer comprising administering ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of an autophagy inhibitor, such as chloroquine. in this regard,
  • therapeutically effective dose of chloroquine may be an initial dose of about 800 mg base followed by an additional 300 mg base and a single dose of 300 mg base on each of two consecutive days. This represents a total dose of 2.5 g chloroquine phosphate or 1.5 g base in three days. In further embodiments, the dose may be about 300 mg base.
  • the dose of chloroquine, or other autophagy inhibitor may be modified as needed by a skilled clinician using dosages known in the art. As would be understood by the skilled person, the autophagy inhibitor may be administered before, at the same time as or after a composition comprising ADI-PEG 20.
  • the dose of ADI-PEG 20 administered for the treatment of pancreatic cancer is between about 50 iU/m 2 and about 8,000 IU/m 2 , and in other embodiments is about 50 IU/m2, about 100 IU/m 2 , 150 IU/m 2 , 200 IU/m 2 , 250 IU/m 2 , 300 IU/m 2 , 350 IU/m 2 , 400 IU/m 2 , 450 IU/m 2 , 500 IU/m 2 , 550 IU/m 2 , 600 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 Mm 2 , 800 IU/m 2 , about 900 IU/m 2 , about 1 ,000 IU/m 2 , 1 ,500 IU/m 2 about 2,000 IU/m 2 , about 2,500 IU/m 2 , about 3,000 IU/m 2 , 3,500 IU/m 2 , 4,000 IU/
  • the dose of ADi-PEG 20 administered for the treatment of pancreatic cancer is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m ⁇ , 8 mg/m 2 , 9 mg/m , 10 mg/m 2 , 15 mg/m , 20 mg/m 2 , 25 rng/rn ⁇ , 30 mg/m 2 , 35 mg/m*, 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 , 80 mg/m 2 , 65 mg/m 2 , 70 mg/m 2 , 75 mg/m 2 , or about 80 mg/m 2 .
  • the present disclosure provides a method of treating pancreatic cancer, wherein the dose of chimeric ADI is doubled and may be increased to 640 I U/m2 per week or more.
  • the chimeric ADI for the treatment of pancreatic cancer is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating pancreatic cancer by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with chloroquine, or other appropriate autophagy inhibitor, wherein the composition comprises a chimeric ADI modified with 5 ⁇ 1 .5 PEG molecules, and in one embodiment, 5 ⁇ 1 .5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of small cell lung cancer by administering a therapeutically effective amount of ADI-PEG 20, optionally in combination with an autophagy inhibitor.
  • the small ceil lung cancer is deficient in ASS, ASL, or both.
  • the present disclosure provides a method of treating small ceil lung cancer comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks;
  • a therapeutically effective dose of chloroquine may be an initial dose of about 800 mg base followed by an additional 300 mg base and a single dose of 300 mg base on each of two consecutive days. This represents a total dose of 2.5 g chloroquine phosphate or 1 .5 g base in three days, in further embodiments, the dose may be about 300 mg base.
  • the dose of chloroquine may be modified as needed by a skilled clinician using dosages known in the art.
  • the autophagy inhibitor may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20.
  • the dose of chimeric ADI-PEG 20 administered for the treatment of small cell lung cancer is between about 50 I U/m 2 and about 8,000 I U/m 2 , and in other embodiments is about 50 ! U/m2, about 100 I U/m 2 , 150
  • I U/m 2 200 I U/m 2 , 250 I U/m 2 , 300 I U/m 2 , 350 I U/m 2 , 400 I U/m 2 , 450 I U/m 2 , 500 I U/m 2 , 550 IU/m 2 , 600 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 IU/m 2 , 800 IU/m 2 , about 900 !U/m 2 , about 1 ,000 IU/m 2 , 1 ,500 IU/m 2 about 2,000 IU/m 2 , about 2,500 IU/m 2 , about 3,000 IU/m 2 , 3,500 IU/m 2 , 4,000 IU/m 2 , 4,500 IU/m 2 , 5,000 IU/m 2 , 5,500 !U/m 2 , 6,000 IU/m 2 , 6,500 IU/m 2 , 7,000 IU/m 2 , 7,500 IU/
  • the dose of chimeric ADI- PEG 20 administered for the treatment of small cell lung cancer is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 , 60 mg/m 2 , 65 mg/m 2 , 70 mg/m 2 , 75 mg/m 2 , or about 80 mg/m 2 .
  • the present disclosure provides a method of treating small cell lung cancer, wherein the dose of chimeric ADi is doubled and may be increased to 640 IU/m2 per week or more.
  • the chimeric AD! for the treatment of small cell lung cancer is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating small ceil lung cancer by administering a composition comprising chimeric ADi - PEG 20 optionally in combination with chloroquine, wherein the composition comprises an chimeric ADI modified with 5 ⁇ 1.5 PEG molecules, and in one embodiment, 5 ⁇ 1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of sarcomas (including but not limited to, metastatic sarcomas) by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with an autophagy inhibitor.
  • the sarcoma is deficient in ASS, ASL, or both, in a further embodiment, the present disclosure provides a method of treating sarcoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of an autophagy inhibitor, such as chloroquine.
  • a therapeutically effective dose of chloroquine may be an initial dose of about 600 mg base followed by an additional 300 mg base and a single dose of 300 mg base on each of two consecutive days. This represents a total dose of 2.5 g chloroquine phosphate or 1.5 g base in three days, in further
  • the dose may be about 300 mg base.
  • the dose of chloroquine may be modified as needed by a skilled clinician using dosages known in the art.
  • the autophagy inhibitor may be administered before, at the same time as or after a composition comprising ADI-PEG 20,
  • the dose of ADI-PEG 20 administered for the treatment of sarcoma is between about 50 IU/m 2 and about 8,000 IU/m 2 , and in other embodiments is about 50 !U/m2, about 100 IU/m 2 , 150 IU/m 2 , 200 IU/m 2 , 250 IU/m 2 , 300 IU/m 2 , 350 IU/m 2 , 400 IU/m 2 , 450 IU/m 2 , 500 !U/m 2 , 550 !U/m 2 , 800 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 IU/m 2 , 800 IU//
  • the dose of ADI-PEG 20 administered for the treatment of sarcoma is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 , 60 mg/m 2 , 65 mg/m 2 , 70 mg/m 2 , 75 mg/m 2 , or about 80 mg/m 2 .
  • the present disclosure provides a method of treating sarcoma, wherein the dose of chimeric ADI is doubled and may be increased to 640 IU/m 2 per week or more, in one particular embodiment the chimeric ADi for the treatment of sarcoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating sarcoma by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with chloroquine, wherein the composition comprises a chimeric ADI modified with 5 ⁇ 1 .5 PEG molecules, and in one embodiment, 5 ⁇ 1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of melanoma by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with docetaxei.
  • the melanoma is deficient in ASS, ASL, or both, in a further embodiment, the present disclosure provides a method of treating melanoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of docetaxei.
  • a therapeutically effective dose of docetaxei may comprise 75mg/m 2 or 100 mg/m 2 administered intravenously over between 30 minutes and 1 hour about every 3 weeks.
  • the dose of docetaxei may be modified depending on disease indication and/or prior treatments, and docetaxei may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20.
  • the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 50 IU/m and about 8,000 IU/m 2 , and in other embodiments is about 50 !U/m2, about 100 iU/m 2 , 150 iU/m 2 , 200 IU/m 2 , 250 IU/m 2 , 300 iU/m 2 , 350 IU/m 2 , 400 IU/m 2 , 450 IU/m 2 , 500 IU/m 2 , 550 IU/m 2 , 600 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 IU/m 2 , 800 IU/m 2 , about 900 IU/m 2 , about 1 ,000 IU/m 2 , 1 ,500 IU/m 2 about 2,000 IU/m 2 , about 2,500 IU/m 2 , about 3,000 IU/m 2 , 3,500 IU/m/m,
  • the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 , 55 mg/m 2 , 60 mg/m 2 , 65 mg/m 2 , 70 mg/m 2 , 75 mg/m 2 , or about 80 mg/m 2 .
  • the present disclosure provides a method of treating melanoma, wherein the dose of chimeric ADI is doubled and may be increased to 640 IU/m2 per week or more.
  • the chimeric ADI for the treatment of melanoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5, PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating melanoma by a composition comprising chimeric ADI-PEG 20, optionally in combination with docetaxei, wherein the composition comprises an chimeric ADI modified with 5 ⁇ 1.5 PEG molecules, and in one embodiment, 5 ⁇ 1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of melanoma by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with cisplatin.
  • the melanoma is deficient in ASS, ASL, or both.
  • the present disclosure provides a method of treating melanoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of cisplatin.
  • a therapeutically effective dose of cisplatin may comprise administration either once per cycle (every 3-4 weeks) at 50-100 mg/m 2 , or daily for 5 days for a total of 100 mg/m 2 per cycle.
  • the dose of cisplatin may be modified depending on disease indication, individual patient, and/or prior treatments, and cisplatin may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20.
  • the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 50 IU/m 2 and about 8,000 IU/m 2 , and in other embodiments is about 50 IU/m2, about 100 IU/m 2 , 150 IU/m 2 , 200 IU/m 2 , 250 IU/m 2 , 300 IU/m 2 , 350 IU/m 2 , 400 IU/m 2 , 450 IU/m 2 , 500 !U/m 2 , 550 IU/m 2 , 600 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 IU/m 2 , 800 IU/m 2 , about 900 IU/m 2 , about 1 ,000 IU/m 2 , 1 ,500 IU/m 2 about 2,000 IU/m 2 , about 2,500 IU/m 2 , about 3,000 IU/m 2 , 3,500 IU/m 2 ,
  • the present disclosure provides a method of treating melanoma, wherein the dose of chimeric ADI is doubled and may be increased to 640 IU/m2 per week or more.
  • the chimeric ADI for the treatment of melanoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5, PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating melanoma by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with cisplatin, wherein the composition comprises a chimeric ADI modified with 5 ⁇ 1.5 PEG molecules, and in one embodiment, 5 ⁇ 1 ,5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of renal ceil carcinoma by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with an mTOR inhibitor, such as but not limited to rapamycin, temsirolimus, everolimus, and ridaforo!imus.
  • the renal cell carcinoma is deficient in ASS, ASL, or both.
  • the present disclosure provides a method of treating renal cell carcinoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks;
  • an mTOR inhibitor such as rapamycin.
  • the dose of rapamycin, or other mTOR inhibitor may be determined as needed by a skilled clinician using dosages known in the art.
  • the mTOR inhibitor may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20.
  • the dose of chimeric ADI-PEG 20 administered for the treatment of renal cell carcinoma is between about 50 IU/m 2 and about 8,000 IU/m 2 , and in other embodiments is about 50 IU/m2, about 100 IU/m 2 , 150 !U/m 2 , 200 IU/m 2 , 250 IU/m 2 , 300 IU/m 2 , 350 IU/m 2 , 400 IU/m 2 , 450 IU/m 2 , 500 IU/m 2 , 550 IU/m 2 , 600 IU/m 2 , 650 IU/m 2 , 700 IU/m 2 , 750 IU/m 2 , 800 IU/m 2 , about 900 IU/m 2 , about 1 ,000 !U/m 2 , 1 ,500 IU/m 2 about 2,000 IU/m 2 , about 2,500 !U/m 2 , about 3,000 IU/m 2 , 3,500 IU/m 2 ,
  • the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 1 mg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , 6 mg/m 2 , 7 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 15 mg/m 2 , 20 mg/m 2 , 25 mg/m 2 , 30 mg/m 2 , 35 mg/m 2 , 40 mg/m 2 , 45 mg/m 2 , 50 mg/m 2 .
  • the present disclosure provides a method of treating renal cell carcinoma, wherein the dose of chimeric ADi is doubled and may be increased to 640 IU/m 2 per week or more.
  • the chimeric ADI for the treatment of renal cell carcinoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI.
  • the present disclosure provides a method of treating renal cell carcinoma by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with rapamycin, or other appropriate mTOR inhibitor, wherein the composition comprises a chimeric ADi modified with 5 ⁇ 1.5 PEG molecules, and in one embodiment, 5 ⁇ 1 ,5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADi (i.e., not modified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
  • the present disclosure also provides methods of treating, ameliorating the symptoms of, or inhibiting the progression of an inflammatory disorder in a patient comprising administering to the patient a composition comprising chimeric ADI (e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20), as described herein, alone or in combination with one or more other therapeutic agents.
  • the present disclosure also provides methods of treating, ameliorating the symptoms of, or inhibiting the progression of an inflammatory bowel disease in a patient comprising administering to the patient a composition comprising chimeric ADI (e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20), as described herein, alone or in combination with one or more other therapeutic agents.
  • the present disclosure provides methods of treating, ameliorating the symptoms of, or inhibiting the progression of Crohn's disease or ulcerative colitis in a patient comprising administering to the patient a composition comprising chimeric ADI (e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20), as described herein, alone or in combination with one or more other therapeutic agents.
  • chimeric ADI e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20
  • the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer in a patient comprising administering to the patient a composition comprising chimeric ADI, and optionally one or more other therapeutic agents, as described herein, wherein the cancer is deficient in ASS, ASL, or both.
  • ASS or ASL deficiency may be a reduction in expression as measured by mRNA expression or protein expression, or may be a reduction in protein activity, and generally comprises a statistically significant reduction in expression or activity as determined by the skilled person.
  • Reduced ASS or ASL expression or activity may be a reduction in expression or activity of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more, as compared to expression or activity in an appropriate control sample known to be cancer free.
  • ASS or ASL expression or activity is reduced by at least twofold as compared to expression or activity in a non-cancer control sample.
  • the reduced expression or activity of ASS or ASL results from methyiation of the ASS or ASL promoter.
  • the reduction in expression or activity of ASS or ASL results from a DNA mutation (e.g., one or more point mutations, small deletions, insertions, and the like) or a chromosomal abnormality resulting in deletion of the gene, in one embodiment, the cancer is ASS or ASL negative, meaning no expression or activity is observed.
  • Reduction in ASS or ASL expression or activity may be measured using any methods known in the art, such as but not limited to, quantitative PCR,
  • enzyme activity assays e.g., assay to measure conversion of cifruliine into argininosuccinafe or conversion of argininosuccinate into arginine and fumarate, and the like.
  • the present invention provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer in a patient comprising administering to the patient a composition comprising chimeric ADI as described herein, wherein the cancer exhibits reduced expression or activity of ASS or ASL, or both, wherein the cancer includes, but is not limited to leukemia (e.g. acute myeloid leukemia and relapsed acute myeloid leukemia), melanoma, sarcomas (including, but not limited to, metastatic sarcomas, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as, but not limited to, hormone refractory prostate cancer), mesothelioma, lymphatic leukemia, chronic
  • gastric cancer including, but not limited to, gastric adenocarcinoma
  • glioma glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney
  • treatment of these ASS-deficient cancers is specifically contemplated herein, with chimeric ADI-PEG alone or in combination with other treatments.
  • the present invention further provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of cancer in a patient comprising administering to the patient a composition comprising chimeric ADI as described herein (e.g. , chimeric ADI- PEG and in particular chimeric ADI-PEG 20), in combination with an autophagy inhibitor.
  • the present invention provides methods for treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a composition comprising chimeric ADI as described herein in combination with autophagy inhibitor wherein the cancer is pancreatic cancer or small ceil lung cancer.
  • the present invention provides methods of treatment where administration of the compositions comprising chimeric AD! described herein depletes arginine in the plasma for at least one month, 2 months, 3 months, 4 months, 5 months, 6 months or longer.
  • This Example describes the generation of artificially engineered chimeric ADI enzymes composed of (1 ) protein with arginine deiminase enzymatic activity, (2) reduced cross reactivity with anti-ADI-PEG 20 antibodies, (3) reduced number of lysine residues, and/or (4) PEG conjugation with chemically stable linkers.
  • ADI Preparation Recombinant chimeric ADI enzymes were cloned, expressed, and purified for testing according to standard protocols, as described, for example, in Gailego et a!., PLOS One, 7(10):e47886, 2012; Monstadt and Holidorf, Biochem. J. 273:739-745, 1990; Joo Noh et a!., Molecules and Cells. 13:137-143, 2002; and Sugimura et a!., Infection and Immunity. 58:2510-2515, 1990. See Table A1 for the amino acid sequences of the chimeric ADI enzymes.
  • Arginine deiminase catalyzes the conversion of L- arginine to L-citruiline and ammonia.
  • the amount of L-citrul!ine can be detected by a colorimetric endpoint assay (see, for example, Knipp and Vasak, Analytical Biochem.
  • ADI ADI expressed as iU/mg of protein.
  • One IU of enzyme activity is defined as the amount of enzyme that produces 1 ⁇ of citru!line per minute at the pH and temperature being tested.
  • Standard assay conditions were performed at 37°C in Physiological HEPES Buffer (PHB) 50 mM HEPES, 180 mM NaCI pH 7.4 (Lang and Zander, Clin Chem Lab Med. 37:563-571 , 1999) plus 0.1 % BSA. All samples and standards were run in duplicate or triplicate where conditions permitted.
  • PLB Physiological HEPES Buffer
  • Km and Kcat values were determined by using a variation of the activity assay described above. As with the activity assay, all reactions were run at 37 ° C in PHB plus 0.1 % BSA. Enzyme concentration, reaction time, and substrate concentration range were adjusted for each of the ADI or ADIr constructs to account for their differences in activity, in general, 2 nM enzyme, 5 minute reaction time, and a 0 - 160 ⁇ arginine was used as starting conditions. When optimizing the conditions, particular attention was paid towards the amount of substrate consumed as a percentage of total substrate added to the reaction. The lower limit of detection is 1 ⁇ of citruiline with the lower limit of quantitation being 2 ⁇ . A citruliine standard curve was run on every plate and used to quantify the citruliine produced by the enzymatic reaction.
  • the results in Table E1 show that the engineered chimeric ADI enzymes described herein have efficient catalytic activity.
  • the catalytic parameters Km and kcat for these enzymes is sufficient to remove arginine and maintain low arginine concentrations in the blood. These parameters are preferably less than 20 ⁇ and greater than 1 sec " 1 , respectively.
  • the pH optimum is around 7.4 so as to maintain efficient catalytic activity in blood.
  • the enzyme stability, as well as that of the covalentiy linked PEG, is such that it should be maintained during long-term storage and patient treatment at 37°C.
  • ADI is composed of two domains, a catalytic domain and an ohelical domain.
  • the present invention is directed in part to engineered, artificial, chimeric, recombinant enzymes with ADI activity. Each is composed of two domains, where each domain is selected from a number of possible species. Domain boundaries are determined by examination of ADI X-ray crystal structures from M. hominis and M. arginini, and extending this to other Mycoplasma ADI enzymes by homology.
  • domains from different species of ADI enzymes can maintain the catalytic activity while changing a number of surface residues. Some of these surface residues form epitopes for anti-ADI-PEG 20 antibodies developed during a patient's treatment with ADi-PEG 20. Their replacement can reduce the antigenicity with respect to anti-ADI-PEG 20 antibodies, therefore reducing anti-ADI-PEG 20 antibody neutralization and clearance of the modified drug. This is shown in Table E2, where two preparations of anti- ADI-PEG 20 antibodies showed less binding to the DS1 , DS2, DS3, and DS4 antigens, compared to M. hominis ADI .
  • the arginini (catalytic domain) - M. arthritidis (a-heiica! domain) chimeras were further modified by replacing surface lysine residues with amino acid residues other than lysine and monitoring ADi activity. Four mutants were made (Table E3) and their ADI activity was determined.
  • Table E4 shows ADI activity of the DS1 (M. arginini - M. arthritidis) enzyme and 4 lysine replacement mutants. Lysine reduction was undertaken to reduce the number of potential pegy!ation sites.
  • the PEG occupancy is generally small at each site. Reducing the number of potential pegylation sites will result in higher PEG occupancies and more complete shielding at each remaining site. This is expected to increase proteolytic protection and reduce immune cross reactivity to affinity matured anti- ADi-PEG 20 antibodies from previous treatments. It will also produce a more uniform drug.
  • the present Examples describes engineered ADl enzymes with excellent ADl activity, with anti-ADi-PEG 20 antibody epitopes removed to reduce antibody neutralization and clearance, and with protection from proteolysis and renal clearance by pegylation.

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Abstract

Provided are chimeric arginine deiminases, including pegylated chimeric arginine deiminases, and related compositions and methods of use thereof, including methods of treating cancer.

Description

ENGINEERED CHIMERIC PEGYLATED ADI AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 1 19(e) to U.S. Application No. 81/954,929, filed on March 18, 2014, which is incorporated by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is TDWG__002__02WO__ST25.txt. The text file is about 200 KB, was created on March 1 1 , 2015, and is being submitted electronically via EFS-Web.
BACKGROUND
Technical Field
The present invention relates generally to engineered ADI, in particular recombinant chimeric ADI proteins engineered to reduce antigenicity. Such engineered chimeric ADI proteins are useful for treating arginine-dependent diseases such as cancer. Description of the Related Art
Amino acid deprivation therapy can be an effective treatment of some forms of cancer. To date, there is one known clinical example relevant to this approach which utilizes asparaginase to lower circulating levels of asparagine and inhibit protein synthesis. This treatment is particuiariy effective for acute lymphoblastic leukemia (Avramis 2005, Viera Pinheiro 2004). Acute lymphoblastic leukemia cells require the amino acid asparagine for growth and proliferation. In contrast, most normal human cells are capable of synthesizing asparagine and are unaffected by asparagine depletion. Therefore, decreasing serum asparagine with asparaginase can selectively kill the cancer ceils without harming the normal cells, tissues, and host. An E. coil derived form of asparaginase has been approved for human use. However, asparaginase is found only in microbes; which makes it highly immunogenic in humans and also has a short serum half-life following injection (Avramis 2005). To make asparaginase a more effective drug, these drawbacks were minimized by formulating the £. coli derived asparaginase with polyethylene glycol (PEG) to reduce the immunogenicity of this enzyme and the associated allergic reactions, in addition, PEG greatly prolongs the circulating half-life of asparaginase, which reduces both the frequency of treatment and the total cost of the therapy. PEG formulated asparaginase is approved for use and is marketed under the trade name Oncaspar© (Oncaspar® 201 1 , Avramis 2005, Viera Pinheiro 2004, Fu 2007, Zeidan 2008).
Arginine is another non-essentiai amino acid for humans and mice (for review see Rogers 1994). In humans, arginine can be synthesized from citruiline in two steps via the Krebs (urea) cycle enzymes argininosuccinate synthetase (ASS, L-citru!iine:L-aspartate iigase [AMP-forming], EC 8.3.4.5) and argininosuccinate lyase (ASL, L-argininosuccinate arginine-!yase, EC 4.3.2.) (Haines 201 1 , Wu 2009, Morris 2006, Husson 2003, Tapiero 2002, Rogers 1994). ASS catalyzes the conversion of citruiline and aspartic acid to argininosuccinate, which is then converted to arginine and fumaric acid by ASL.
ADi-PEG 20 treatment requires multiple doses over a period of time. After a number of treatments, anti-ADI-PEG 20 antibodies can develop that may limit its continued effectiveness. Therefore, there is a need in the art for ADI that is engineered to improve and extend the efficacy of arginine depletion therapy.
References: Oncaspar FDA Label, Revised 7, 2008; downloaded from FDA website on April 5, 201 1 ; Avramis VI, Panosyan EH. 2005. Clin Pharmacokinet 44:387-393; Fu CH, Sakamoto KM. 2007. Expert Opin Pharmacother 8:1977-1984; Haines RJ, et ai. 201 1 . Int J Biochern Mol Biol 2:8-23; Husson A, et al. 2003. Eur J Biochem 270:1887-1899; Morris SM Jr. 2006. Am J Clin Nutr 83(Suppl):598S-512S; Viera Pinheiro JP, Boos J. 2004. Br J Haematol 125: 1 17-127; Wu G, et al. 2009. Amino Acids 37:153-168; Zeidan A, et al. 2008. Expert Opin Biol Ther 9:1 1 1 -1 19; Rogers QR. Special Publication 86, Agriculture Experiment Station, University of Illinois, April 4-5, 1994:9-21 ; Tapiero H, athe G, Couvreur P, Tew KD (2002) I. Arginine. Biomed Pharmacother 56: 439-445; Wheatley DN (2004) Anticancer Drugs 15(9): 825-833; Feun LG, et al, British Journal of Cancer, 2012, 106, 1481-1485; Dillon BJ, et al, Cancer, 2004, 100(4), 826-33.
BRIEF SUMMARY
One aspect of the present invention provides a recombinant chimeric arginine deiminase (ADI) comprising a catalytic domain of an ADI protein derived from a first microorganism and an a-helical domain of an ADI protein derived from a second
microorganism. In one embodiment, the first microorganism is selected from the genera Mycoplasma, Clostridium, Bacillus, Borrelia, Enterococcus, Streptococcus, Lactobacillus, and Giardia, In a further embodiment, the first microorganism is selected from the group consisting of Mycoplasma pneumonia, Mycoplasma hominis, Mycoplasma arginini,
Mycoplasma arthritidis, Streptococcus pyogenes, streptococcus pneumonia, Borrelia burgdorferi, Borrelia afzeiii, Giardia intestinaiis, Clostridium perfringens, Bacillus
iicheniformis, and Enterococcus faecaiis. In yet another embodiment, the first microorganism is selected from the group consisting of M. arginini, M. arthritidis, M. hominis, Mycoplasma pneumonia, Mycoplasma phocicerebrale, Mycoplasma orale, Mycoplasma galeae,
Mycoplasma phocidae, Mycoplasma columbinum, Mycoplasma iowae, Mycoplasma crocodyli, Mycoplasma fermentans, Mycoplasma gaiiinarum, Mycoplasma iners,
Mycoplasma penetrans, Mycoplasma gallisepticum, Mycoplasma alligatoris, Mycoplasma mobile, and Mycoplasma capricolum. in one embodiment, the second microorganism optionally differs from the first microorganism and is selected from the genera Mycoplasma, Clostridium, Bacillus, Borreiia, Enterococcus, Streptococcus, Lactobacillus, and Giardia. In another embodiment, the second microorganism optionally differs from the first
microorganism and is selected from the group consisting of Mycoplasma pneumonia, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Streptococcus pyogenes, streptococcus pneumonia, Borreiia burgdorferi, Borreiia afzelii, Giardia intestinails, Clostridium perfnngens, Bacillus licneniformis, and Enterococcus faecaiis. in yet another embodiment, the second microorganism optionally differs from the first
microorganism and is selected from the group consisting of M. arginini, M. arthritidis, M, hominis, Mycoplasma pneumonia, Mycoplasma phocicerebrale, Mycoplasma orale,
Mycoplasma gateae, Mycoplasma phocidae, Mycoplasma columbinum, Mycoplasma lowae, Mycoplasma crocodyli, Mycoplasma fermentans, Mycoplasma gaiiinarum, Mycoplasma iners, Mycoplasma penetrans, Mycoplasma gallisepticum, Mycoplasma alligatoris,
Mycoplasma mobile, and Mycoplasma capricolum.
In one embodiment, the first microorganism is selected from the group consisting of Mycoplasma gaiiinarum, Mycoplasma iners, and Mycoplasma columbinum and the second microorganism is selected from the group consisting of Mycoplasma gaiiinarum, Mycoplasma iners, and Mycoplasma columbinum, wherein the first and second
microorganism are optionally different microorganisms.
in a further embodiment, the first microorganism is M. arginini and the second microorganism is M. arthritidis, and in other specific embodiments, the first microorganism is M. arginini and the second microorganism is M. hominis or the first microorganism is M. arthritidis and the second microorganism is M. arginini. in certain embodiments, the first microorganism is M. gateae and the second microorganism is M. arthritidis. in certain embodiments, the first microorganism is M. gateae and the second microorganism is M. columbinum. In some embodiments, the first microorganism is M. gateae and the second microorganism is M. phocicerebrale. in some embodiments, the first microorganism is M. gateae and the second microorganism is M. phocidae. In particular embodiments, the first microorganism is M. phocicerebrale and the second microorganism is M. arginini. In certain embodiments, the first microorganism is M. phocicerebrale and the second microorganism is M. gateae. in specific embodiments, the first microorganism is M. phocicerebrale and the second microorganism is M. phocicerebrale. In certain embodiments, the first microorganism is M. phocidae and the second microorganism is M. arginini. In some embodiments, the first microorganism is M. phocidae and the second microorganism is M. arthritidis. In certain embodiments, the first microorganism is M. phocidae and the second microorganism is M. coiumhinum. In particular embodiments, the first microorganism is M. phocidae and the second microorganism is M. gateae. In certain embodiments, the first microorganism is M. phocidae and the second microorganism is M. phocicerebrale. In some embodiments, the first microorganism is M. gal!inarum and the second microorganism is M. coiumhinum. In certain embodiments, the first microorganism is M, gaiiinarum and the second
microorganism is M. iners, In some embodiments, the first microorganism is M. iners and the second microorganism is M. columbinum. In certain embodiments, the first microorganism is iners and the second microorganism is M. gaiiinarum.
illustrative recombinant chimeric ADI molecules comprise, consist, or consist essentially of the amino acid sequence set forth in any one of SEQ ID NOs:4-13 or 22-59 or a variant thereof having at least 80% or 90% sequence identity to any of SEQ ID NOs:4-13 or 22-59.
in certain embodiments of the recombinant chimeric ADi described herein, the recombinant chimeric ADi has been modified to remove at least one pegylation site. In another embodiment of the recombinant chimeric ADI described herein, at least one lysine residue has been modified by an amino acid substitution. In certain embodiments of the recombinant chimeric ADI described herein, at least 5 lysine residues have been modified by an amino acid substitution, at least 10 lysine residues have been modified by an amino acid substitution, at least 15 lysine residues have been modified by an amino acid substitution, or at least 20 lysine residues have been modified by an amino acid substitution. Illustrative recombinant chimeric ADI molecules as described herein comprise the amino acid sequence set forth in any one of SEQ I D NOs: 10-13.
in another embodiment of the recombinant chimeric ADI described herein, the ADI is covalently bonded via a biocompatible linker to polyethylene glycol. In this regard, the arginine deiminase may be covalently bonded to more than one polyethylene glycol molecule and in certain embodiments may be covalently bonded to about 1 to about 10 polyethylene glycol molecules and in one specific embodiment, to 5±3 PEG molecules. The PEG molecules covalently bonded to the ADI as described herein may be straight chain or branch chain PEG molecules and may have a total weight average molecular weight of from about 1 ,000 to about 40,000 and in one embodiment, from about 10,000 to about 30,000. in certain embodiments of the recombinant chimeric AD! described herein, the biocompatible linker comprises a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a hisfidine group, a methylene group, or any combinations thereof. In one embodiment, the source of the succinyl group is succinimidyl succinate.
Other aspects of the invention provide a polynucleotide encoding the recombinant chimeric AD I described herein, vectors comprising the polynucleotide and isolated host cells comprising such vectors.
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences,
po!yadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et a!., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into ceils and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubei et a!. eds., John Wiley & Sons, 1992, or subsequent updates thereto.
As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non- naturaliy occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. Such
polynucleotides are commonly referred to as "codon-optimized." Any of the polynucleotides described herein may be utilized in a codon-optimized form. In certain embodiments, a polynucleotide can be codon optimized for use in specific bacteria such as E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown ef a/., Protein Expr Purif. 59:94-102, 2008).
Systems for cloning and expression of a protein in a variety of different host cells are well known. Suitable host cells include mammalian cells, bacteria, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, HEK-293 cells, human fibrosarcoma cell line HT-1080 (see, e.g.,
Moran, Nat. Biotechnol. 28:1 139-40, 2010), NSO mouse melanoma cells and many others. Additional examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et ai., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney ceils (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney ceils (MDCK, ATCC CCL 34); buffalo rat liver ceils (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TR1 cells (Mather ef ai, Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et ai, PNAS USA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For a review of certain mammalian host ceil lines suitable for polypeptide production, see, e.g., Yazaki and Wu, Methods In Molecular Biology, Vol. 248 (B, K,C Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268. Certain preferred mammalian ceil expression systems include CHO and HEK293-celi based expression systems. Mammalian expression systems can utilize attached cell lines, for example, in T- fiasks, roller bottles, or cell factories, or suspension cultures, for example, in 1 L and 5L spinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50L and 100/200L WAVE bioreactors, among others known in the art.
A common bacterial host is £. coii. The expression of proteins in prokaryotic cells such as E. coii is well established in the art. For a review, see for example Piuckthun, A. Bio/Technology. 9:545-551 (1991 ). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for recombinant production of polypeptides (see Ref, Curr. Opinion Biotech. 4:573-576, 1993; and Trill et ai., Curr. Opinion Biotech. 6:553-560, 1995). In specific embodiments, protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series). These and related embodiments may utilize the expression host strain BL21 (DE3), a X.DE3 lysogen of BL21 that supports T7~mediated expression and is deficient in ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coii, such as Rosetta" (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling may also be improved using reagents such as Benzonase® nuclease and BugBuster® Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems. Media of this type (e.g., Overnight Express™ Autoinduction System) gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG. Particular embodiments employ hexahistidine tags (such as His»Tag® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. In certain aspects, however, clinical grade proteins can be isolated from £. coli inclusion bodies, with or without the use of affinity tags (see., e.g., Shimp ef a/., Protein Expr Punt. 50:58-67, 2008). As a further example, certain embodiments may employ a cold-shock induced E. co!i high-yield production system, because over-expression of proteins in
Escherichia coii at low temperature improves their solubility and stability (see, e.g., Qing ef a/., Nature Biotechnology. 22:877-882, 2004).
in addition, a host celi strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, post- transiationai modifications such as acetylation, carboxylation, glycosyiation, phosphorylation, lipidation, and acylation. Post-franslational processing, which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the protein of interest.
For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, ceil lines that stably express a
polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which, successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the celi type. Transient production, such as by transient transfection or infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include HEK293 and CHO-based systems.
Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from ceil culture. Certain specific embodiments utilize serum free cell expression systems. Examples include HE 293 ceils and CHO cells that can grow on serum free medium (see, e.g., Rosser ef a/., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent number 6,210,922). The proiein(s) produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art, Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) {e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography {e.g., Q, 8), size exclusion
chromatography, salt gradients, affinity purification {e.g., Ni, Co, FLAG, maltose, glutathione, protein A G), gel filtration, reverse-phase, ceramic HyperD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS-PAGE {e.g., coomassie, silver stain), immunoblot,
Bradford, and ELISA, which may be utilized during any step of the production or purification process, typically to measure the purity of the protein composition.
Also included are methods of concentrating recombinantly produced proteins, e.g., chimeric ADi proteins. Examples include iyophiiization, which is typically employed when the solution contains few soluble components other than the protein of interest.
Lyophilization is often performed after HPLC run, and can remove most or all volatile components from the mixture. Also included are ultrafiltration techniques, which typically employ one or more selective permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.
in certain embodiments, the chimeric ADi proteins have a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain therapeutic compositions, the chimeric ADI proteins have a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the chimeric ADi proteins have a purity of at least about 97% or 98% or 99%. in other embodiments, such as when being used as reference or research reagents, proteins can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.
in certain embodiments, the compositions described here are about substantially endotoxin free, including, for example, about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free. The presence of endotoxins can be detected according to routine techniques in the art, as described herein, in specific embodiments, the chimeric ADi proteins are made from a eukaryotic ceil such as a mammalian or human ceil in substantially serum free media. Another aspect of the invention provides a composition comprising one or more of the recombinant chimeric ADi described herein and a physiologically acceptable carrier. In one embodiment, the composition may further comprise an autophagy modulator, Autophagy modulators include, but are not limited to chloroquine, 3-methyiadenine, hydroxychloroquine, bafiiomycin A1 , 5-amino-4-imidazoie carboxamide riboside (AICAR), okadaic acid, N6-mercaptopurine riboside, vinblastine, wortmannin, rapamycin, everoiimus, metformin, perifosine, resveratrol, and tamoxifen. In another embodiment, the compositions comprising the recombinant chimeric ADI described herein may further comprise a chemotherapeutic agent, such as but not limited to docetaxei, carbopiatin,
cyclophosphamide, gemcitabine, cisplatin, sorafenib, sunitinib or everoiimus, or
combinations thereof.
Yet another aspect of the present invention provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising one or more of the recombinant chimeric ADi described herein and a
physiologically acceptable carrier, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer. In this regard, the cancer can include, but is not limited to melanoma, pancreatic cancer, prostate cancer, small cell lung cancer,
mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 is the amino acid sequence of wild type M. ho inis ADi.
SEQ ID NO:2 is the amino acid sequence of wild type M. arginini ADI.
SEQ ID NO:3 is the amino acid sequence of wild type M. arthritidis ADi.
SEQ ID NO:4 is the amino acid sequence of the DS1 recombinant chimeric
ADI protein.
SEQ ID NO:5 is the amino acid sequence of the DS2 recombinant chimeric
ADI protein.
SEQ ID NO:8 is the amino acid sequence of the DS3 recombinant chimeric ADI protein. SEQ ID NO:7 is the amino acid sequence of the DS4 recombinant chimeric
ADI protein.
SEQ ID NQ:8 is the amino acid sequence of a recombinant chimeric AD! derived from M. hominis Phoenix (see SEQ ID NO: 14; plus lysine substitutions) (catalytic domain) and M. arginini (a-helical domain).
SEQ ID NO:9 is the amino acid sequence of a recombinant chimeric ADi derived from M. hominis Phoenix (see SEQ ID NO: 14) (catalytic domain) and M. arthntidis (a-helicai domain).
SEQ ID NO: 10 is the amino acid sequence of the DS1 -1 lysine reduction mutant of the recombinant chimeric ADi protein (see Table E3).
SEQ ID NO:1 1 is the amino acid sequence of the DS1 -2 lysine reduction mutant of the recombinant chimeric ADI protein (see Table E3).
SEQ ID NO: 12 is the amino acid sequence of the DS1 -3 lysine reduction mutant of the recombinant chimeric ADi protein (see Table E3).
SEQ ID NO: 13 is the amino acid sequence of the DS1 -4 lysine reduction mutant of the recombinant chimeric ADi protein (see Table E3).
SEQ ID NO:14 is the amino acid sequence of the ADi Phoenix sequence. This ADI sequence is identical to M hominis ADI except for K1 12E and P210S substitutions.
SEQ ID NO: 15 is the amino acid sequence of M. aiiigatoris ADI. SEQ ID NO:16 is the amino acid sequence of M. colombinum ADi.
SEQ ID NO:17 is the amino acid sequence of gailinarum ADi.
SEQ ID NO: 18 is the amino acid sequence of M, galea ADI.
SEQ ID NO: 19 is the amino acid sequence of M, iners ADL
SEQ ID NO:20 is the amino acid sequence of M, phocicerabra!e ADL SEQ ID NO:21 is the amino acid sequence of M. phocidae ADI.
SEQ ID NOs:22-59 are the amino acid sequences of chimeric ADI proteins.
DETAILED DESCRIPTION
The present invention relates generally to chimeric ADi proteins, e.g., engineered to have reduced antigenicity as compared with corresponding wild type ADI molecules. The present invention also relates to methods of treating cancer and other disorders with chimeric ADI, and in particular chimeric ADI-PEG 20.
Normal cells do not require arginine for growth, since they can synthesize arginine from citrulline in a two-step process catalyzed by ASS and ASL. In contrast, certain cancers do not express ASS. Certain cancers do not express ASL, and other cancers may have diminished expression of, or may not express ASS and/or ASL. Therefore, these cancers are auxotrophic for arginine. This metabolic difference may be capitalized upon to develop a safe and effective therapy to treat these forms of cancer, ADI catalyzes the conversion of arginine to citrulline via the arginine dihydrolase pathway, and may thus be used to eliminate arginine.
The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in immunology, John Wiley & Sons, New York, N.Y.(2009);
Ausubel et a/., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Moiecular Cloning: A Laboratory Manual (3rd Edition, 2001 ); Maniatis et ai Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N, Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Ceil Culture (R. Freshney, ed., 1986); Perbai, A Practical Guide to Molecular Cloning (1984) and other like references.
As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
By "about" is meant a quantify, Ievel, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 8, 5, 4, 3, 2 or 1 % to a reference quantity, Ievel, value, number, frequency, percentage, dimension, size, amount, weight or length.
By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance Ievel, then the null hypothesis is rejected, in simple cases, the significance level is defined at a p-value of 0.05 or less.
Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, !ipofection).
Enzymatic reactions and purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
"Patient" refers to an animal, in certain embodiments a mammal, and in a specific embodiment, a human.
"Biocompatible" refers to materials or compounds which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.
The term "reference sequence" refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.
Throughout the present disclosure, the following abbreviations may be used:
PEG, polyethylene glycol; ADI, arginine deiminase; SS, succinimidyl succinate; SSA, succinimidyl succinimide; SPA, succinimidyl propionate; NHS, N-hydroxy-succinimide; ASS1 or ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase.
in certain embodiments, the chimeric ADI enzymes as described herein are compared to the benchmark ADI-PEG 20 molecule derived from hominis. As used herein, "ADI-PEG 20" refers to the ADI molecule known in the art and described for example in US6183738; US8635462; Ascierto PA, et ai. (2005) Pegyiated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and Π studies. J Clin Oncol 23(30): 7660- 7668; Izzo F, et ai (2004) Pegyiated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase i/ll studies. J Clin Oncol 22(10): 1815-1822; Holtsberg FW, et ai. (2002), Polyethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties. J Control Release 80(1- 3): 259-271 ; Kelly ef a/., (2012) British Journal of Cancer 106, 324 - 332. As would be recognized by the skilled artisan, this molecule is a pegylated (PEG 20,000) ADI enzyme derived from M. hominis, and has two substitutions (K1 12E; P210S) relative to the wild type M. hominis ADI enzyme.
The terms "polypeptide," "protein" and "peptide" and "enzyme" are used interchangeably and mean a polymer of amino acids not limited to any particular length. The terms do not exclude modifications such as myristoyiation, sulfation, glycosyiation, phosphorylation and addition or deletion of signal sequences. The terms "polypeptide" or "protein" or "enzyme" mean one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant ceils, or genetically-engineered or recombinant ceils, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass the chimeric ADI enzymes of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of the chimeric ADI enzymes. In certain embodiments, the polypeptide is a "recombinant" polypeptide, produced by recombinant ceil that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.
The term "isolated protein" referred to herein means that a subject protein (1 ) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovaient interaction) with portions of a protein with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or noncovaient interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise). in the present invention, a chimeric ADI or a polynucleotide encoding a chimeric ADI may be derived, cloned or produced from any source, including, for example, microorganisms, recombinant biotechnology or any combination thereof. For example, arginine deiminase may be cloned from microorganisms of the genera Mycoplasma.
Clostridium, Bacillus, Borreiia, Enterococcus, Streptococcus, Lactobacillus, Giardia. in certain embodiments, arginine deiminase is cloned from Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma phocicerebraie, Mycoplasma oraie, Mycoplasma galeae, Mycoplasma phocidae,
Mycoplasma columbinum, Mycoplasma iowae, Mycoplasma crocodyii, Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma galiisepticum, Mycoplasma gailinarum. Mycoplasma iners, Mycoplasma aliigatoris, Mycoplasma mobile, and Mycoplasma capricolum, Steptococcus pyogenes, Steptococcus pneumoniae, Borreiia burgdorferi, Borreiia afzeiii, Giardia intestinalis, Clostridium perfringens, Bacillus licheniformis,
Enterococcus faecalis, Lactobacillus sake, or any combination thereof. The amino acid sequences of certain of these arginine deiminases is provided in TaWe A1 bdow.
Figure imgf000015_0001
DPMPNLYFTRDPFASVGNGISLHNMKYQTRKRETIFAQFIFKYNKD YKTTPHWFDRFDHGS IEGGDVFVYTKDTLVIGI SERTTKEAVLNIAKK
1KANTDSKFKKIVAINVPPMPNLMHLDT ITMVDHDKFLYSP MMKSL KFWLIDLSKEIKMVELEESLSNMLEAI IGKKPILIPIAGKNASQLDID IΞTHFDGT YLT IAPG VVGYSR KLTQKALEDAGVKVLSFDGMQLSL GMGSARCMSMPLVREDIK
M. colombinuni MSKI VYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAI 16
KEHKGFLKI LQDKGIKVIQLSDL AETYTYHATQKEREAFIEK LDEA EPALTKDLRAKVKSYVLSKEGTPVAMVRT'MMAGVSKQELNVESET'ELV VDP P LYFTRDPFASAGNGISL MKYVTRKRET FAEFIFATHPDY KT PHWFDRLDEG IEGGDVFIYNKDTLVIGVSERTNKEAILTI KKI KN EAKFKKIVAI VPPMPNLMHLDT LTMVDKDKFLYSP LSVLK WE I DLSKE IEMVETMKPLADVLES I IGVKPVLI PIAGKGA QLDI DI ETHFDGT YLTIAPGVVVGYSRNIKTEAALRAAGVTVLSFEGNQLSLG GSARCMSMPLVREDVK
M. gallinarum MSKTRVYSEIGNLKKVLVHTPGDEIRRISPSRLEELLFSAVLEPNAAI 17
EEHKRFVKLLF.DRGIQAIOLSDLVAETYVKYATAEQKAAFIEKYLDEA TPALSAENRERAKKYILSLEMQPVKMIRT MAGLSKYELNVES IELI IDPMPNLYFTRDPFASAG GISL NMKYWRKRETIFAEFIFAIHPEY KETPHWFDRLDHGSIEGGDVFVYNKDTLVIGVSERT KEAI ITIAKHI QDNKEAEFKKIVAINVPPMPNLMHLDTWLTMVDKNKFIYSPNMLSVLK IWE I DLAKPIEMVESNKSLTEVLES 11GEKPI LI PIAGEGASQLDI DI ETHFDGT YL PGWVGYSR EKTEKALKAAGI VLSFEGNQLSLG MGSARCMSMPLVREDVK
M. gatea MSVFDSKFNGIHVYSETGELESVLVHEPGRE I DYI TPARLDELLFS I 18
LESHDARKEHKLFVSELKANDI WELTDLVTETYDLASQEAKDNLIE EFLEDSEPVLTEELKSWRTYLKSIKS RELIQMMMAGITKYDLGIEA DKELIVDPMPNLYFTRDPFASVGNGVTIHYMRYKVRQRETLFSRFVFS NHPKLVNTPWYYDPSLKLSIEGGDVFIYNNNTLWGVSERTDLETVTL LAKMIV KECEFKR VAT VpKWT LMHLD WLTMLDKDKFLYS PI NDV b'Kfc'WDYDLVNGGEEPQPVENGLPLEGLLES 1 INKKPILl PIAGEG ASQI DIERETHFDGTNYLA IRPGVVIGYSRNEKTNAALEAAGIKVLPF HGNQLSLGMGNARCMSMPLSRKDVKW
M. iriers MSKI VYSEIGVLKEVLVHTPGDEIRRIAPSRLDELLFSAILEPSAAI 19
QEHKSFLKILQDRGIKT IQLSDLVAETΥΚΗΥΑΒΕΑΞΚΕΑΒΊEKYLDEA TPVLSKDMRAKVKN I LSMQGEP KMVRTMMAGVSKQELN ESEVELI VDPMPNLYFTRDPFASAGNGISLNNMKYWRKRETIFAEFIFSIHPEY KKTPH FDRLDNGSIEGGDVFIY KDTLVIGVSERTN EAIITIA HI QDNKEAQFKKIVAI VPPMPNLMHLDTWLTMVDKNKFLYSPNMLSVLK VWEIDLSKPIEMVETNKPLAEVLES 11GEKPILI PIAGKDATQLDIDI ETHFDGTNYLTIAPGWVGYSRNVKTEAALRAAGVTVLSFEGNQLSLG MGSARCMSMPLVREDVK
M. MSVFDSKFNGIHVYSΞ IGELETVLVHEPGRE I D I TPARLDELLFSAI 20 phocicerabrale LESHDARKEHQSFVKQLKDNGINWELTDLVAETFDLASKEEQEKLIE
EFLEDSEPVLSEAHKTAVRKFLTSRKSTREMVEFMMAGITKYDLGIEA DHELIVDPMPNLYFTRDPFASVGNGVTIHYMRYKVRQRETLFSRFVFS NHPKLVKTPWYYDPAMKMSIEGGDVFIYNNDTL GVSERTDLETITL LAK IKANKEVEFKRIVAINVPK ' NLMHLDTWLTMLDKDKFLYS PIA NDVFKFWDYDLVNGGAEPQPKENGLPLEGLLQSI INKKPVLIPIAGNN ASHIDIERETHFDGTNYLAIKPGVVIGYARNEKTNAALAAAGIKVLPF HGNQLSLGMGNARCMSMPLSRKDVKW
M. phocidae MSVFDSKFNGIHVYSEIGELQTVLVHEPGREIEYITPARLDELLFSAI 21
LESHDARI<E,KQEFVAELK1NNINVVELTDLVSETYDMVSKEKQEKLIE EFLEDSEPVLSEEHKGLVRKFLKSLKSSKELIQYMMAGITKI-IDLNIEA DHELIVDPMPNLYFTRDPFASVGNGVTIHYMRYKVRQRETLFSRFIFA NHPKLMMTPLYYNPDMKLS IEGGDVFVYN ΞTLVVGVSERTDLDT I TL LAKNTKANKEREFKRIVAINVPKWTNLMHLDTWLTMLDKDKFLYSPIA NDVFKh'WDYDLVMGGDEPQPKV GLPLEKLLES i INKKPIL1 PIAGTS ASNIDVERETHFDGTNYLAIAPG IGYSRNVKTNEALEAAGIKVLPF KGNQLSLGMGNARCMSMPLSRKDVKW Thus, in some embodiments, the ADI used in a chimeric ADI may comprise the amino acid sequence from Table A1 (any one of SEQ ID NOs: 1-3 or 15-21 ), or a variant thereof having ADI activity (e.g., able to metabolize arginine into citrulline and ammonia) or a fragment thereof having ADi activity, or engineered chimeras thereof having ADi activity.
The sequences of exemplary chimeric ADi are provided in Table A2 below.
Table A2. Chimeric ADI
Name Sequence SEQ ID
NO :
MSVFDSKFKGIHVYSEIGELESVLVHEPGRE I DY I TPARLDELLF'SAILESH 4 DARKEHKQFVAELKANDINWELVDLIVETYDLASKEAKE LLEEFLDDSVP VLSDEHRATVKKFLQSQKSTRSLVEYMIAGITKHDLKIESDLELIVDPMPNL YFTRDPFASVGNGVTIHYMRYKVRQRETLFSREVFSNHPKLINTP YYDPSL
DSl
KLSIEGGDVFIY NDTLWGVSERTDLQTVTLLAKNIVA KESEFKRIVAIN VPKWT LMHLDTWLTMLDKDKFLYSPIANDVFKFWDYDLVNGGAE QPVENG LPLEGLLQSIINKKPVLIPIAGEGASQMEIERETHFDGT YLAIRPGWIGY SR EKTNAALEAAGIKYLPFHGNQLSLGMGNARCMSMPLSRKDVK
MSVFDSKFKGIHVYSETGELESVLVHEPGRE I DYI TPARLDELLFSAILESH 5 DAR EHKQFVAELKANDINWELTDLVAETYDLASRAAKEEFIETFLEETVP VLTEANREAVRAFLLSKPTHEMVEFMMSGITKYELGVESE ELIVDPMPNLY FTRDPFASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLINTP YYDPSLK
DS2
LSIEGGDVFIYNNDTLWGVSERTDLQTVTLLAK IVA KESEFKRIVAINV PKWTNLMHLDTWLTMLDKDKFLYSPIANDVFKFWDYDLVNGGAEPQPVENGL PLEGLLQS11 K PVLIPIAGEGASQMEIERETHFDGTNYLAIRPGWIGYS RNEKTNAALEAAGIKVLPFHGNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELETVLVH.EPGKE IDYITPARLDELLFSAILESH 6 DARKEHKEFVAELKKRGINWELIDLVAETYDLASQEAKDKLIEEFLEDSEP VLSEEHKWVR FL AKKTSRELVEIMMAGI YDLGIEADHELIVDPMPNL YFTRDPFASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLV TPWYYDPAE
DS3
GLTIEGGDVFI YN DTLWGVSERTDLQTITLLAK IKA KESEFKRIVAIN VPKWTNLMHLDTWLTMLDKDK LYSPIANDVFKFWDYDLV GGDAPQPVDNG LPLEDLLKSI IGKKPTLI PIAGAGASQIDIERETHFDGTNYLAVAPGIVIGY ARNEKT AALEAAGITVLP RG QLSLGMGNARCMSM LSRKDVKW
MSVFDSKFKGIHVYSEIGELETVLVHEPGKE I DYITPARLDELLFSAILESH 7 DARKEHKEFVAEL KRGI WELTDLVAETYDLASRAA EEFIETFLEETVP VLTEANREAVRAFLLSKPTHEMVEFMMSGITKYELGVESENELIVDPMPNLY FTRDPFASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVKTP YYDPAEG
DS4
IJT IEGGDVFIYNNDTLWGVSERTDLQTITLLAKMIKANKESEFKRIVAI V PKWTNLMHLDT LTMLDKDKFLYSPIANDVFKFWDYDLVNGGDAPQPVDMGL PLEDLLKSI IGKKPTLI PI GAGASQI DIERETHFDGTNYLAVAPG IVIGYA RNEKTNAALEAAGI VLPFRGNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 22 KEHKQFVAELKANDINVVELDELVAQTYDQVDQKIKDEFIDQWLQEAKPVLNDQL KKLVKNYLLKSQKEFSTKKMVRIMMAGIDKKEINIDLDRDLVVDPMPNLYFTRDP FASVG GVTIHYMRYKVRQRETLFSRFVFS HPKLINTPWYYDPSLKLSIEGGDV
C2DS1
FIYNNDTLVVGVSERTDLOTVTLLAKNIVANKESEFKRIVAINVPKWTNLMHLDT LTMLDKDKFLYSPiANDVh'KFWDYDLV GGAEPQPVENGLPLEGLLQSI INKKP
VLIPIAGEGASQMEIERETKFDGTNYLAIRPGVVIGYSRNEKTNAALEAAGIKVL PFHGNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELESVLVHE GREIDYITPARLDELLFSAILESHDAR 23 KEKKQFVAELKANDINVVELVDLIVETYDLASKEAKEKLLEEFLDDSVPVLSDEH
C2DS3
RAT KKFLQSQKSTRSLVEYMIAGITKHDLKIESDLELI DPMPNLYFTRDPFAS VG GVTIHYMRYKVRQRETLFSRFVFS HPKLINTPWYYDPSLKLSIEGGDVFIY NDTLWGVSERTDLQTVTLLAKNIVANKESEFKRIVATNVPKWTNLMHLDTWLT MLDKDKFLYSPIANDVFKFWDYDLVNGGAEPQPVENGLPLEG-LLQSI INKKPVLI PIAGEGASQMETERETHFDGTNYLAIRPGVVIGYSR EKTNAALEAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELBSVLVHEPGREIDYITPARLDELLFSAILESHDAR 24 KEHKQFVAELKANDIN ELSDLVAETYTYI-IATQKEREAFIEKWLDEAEPALTKD LRAKVKSYVLSKEGTPVAMVRTMMAGVSKQELNVESETELWDPMPNLyFTRDPF ASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLINTPWYYDPSLKLSIEGGDVF
C2DS4
TYNNDTLWGVSERTDLQ VTLLAKNTVANKESEFKRIVAINVPKWTNLMHLDTW LTMLDKDKFLYSPiA DVFKF DYjijVNGGAEPQPVENGLPijEGLLQSI I KKPV LIPIAGEGASQMEIERETI-IFDGT YLAIRPGVVIGYSRNEKT AALEAAGIKVLP FHGNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 25 KEHKQFVAELKA DI WELTDLVTETYDLASQEAKDNLIEEFLEDSEPVLTEEL KSVVRTYLKSIKSTRELIQMMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS
C2DS5 VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLINTP YYDPSLKLSIEGGDVFIY
NDTLWGVSERTDLQTVTLLAKNIVANKESEFKRIVATNVPKWTNLMHLDTWLT MLDKDKh'LYSPTANDVFKFWDYDLVNGGAEPQPVENGLPLEGLLQSI INKKPVLI PIAGEGASQMEIERETHFDGTNYLAIRPGWIGYSRNEKTNAALEAAGI VLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 26 KEHKQFVAELKANDIN ELTDLVAETFDLASKEEQEKLIEEFLEDSEPVLSEAH KTAVRKFLTSRKSTREMVEFMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS VGNGV IHY RYKVRQRETLFSRFVFSNHPKLIN P YYDPSLKLSIEGGDVFIY
C2DS6
DTLWGVSERTDLQTVTLLAK IVANKESEFKRIVAI VPK TNLMHLDTWLT MLDKDKFLYSPIANDVFKFWDYDLVNGGAEPQPVENGLPLEGLLQSi INKKPVLI. PIAGEGASQMSIERETHFDGTNYLAIRPGVVIGYSRNEK NAALEAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFKGIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 27 KEKKQFVAELKANDINVVELTDLVSETYDMVSKEKQEKLIEEFLEDSEPVLSEEH KGLVRKFLKSLKSSKELIQY MAGITKHDLNIEADHELIVDPMPNLYFTRDPFAS
C2DS7 VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLINTPWYYDPSLKLSIEGGDVFIY
N DTLWGVSERTDLQTVTLLAKNIVANKESEFKRIVAI VP TNLMHLDTWLT MLDKDKi'LYSPIANDVJ;' FvJDYDLVNGGAEPQPVENGbPLEGLLQSII KKPVLI PIAGEGASQMEIERETHFDGTNYLAIRPGVVIGYSRNEKTNAALEAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW SKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 28 KILQDKGIKVIQLDELVA iTYDQVDQKIKDEFIDQWLQEAKPVLNDQLKKLVKNY LLKSQKEFSTKKMVRIMMAGIDKKEINIDLDRDLVVDPMPNLYFTRDPFASAGNG ISLN KYVTRKRETIFAEFIFATHPDYKTTPH FDRLDEGNIEGGDVFIYNKDT
C4DS1
LVIGVSERTNKEAILTIAKKIK KEAKFKKIVAI VPPMPNLMHLDTWLTMVDK DKFLYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESI IGVKPVLIPIAGKGA TQLDIDIETHFDGTNYLTIAPGVVVGYSRNIKTEAALRAAGVTVLSFEGNQLSLG MGSARCMSMPLVREDVK
MSKINVYSEIGELKEVLVI-ITPGDEIRRISPSRLDELLFSAILEP EAIKEI-IKGFL 29 KILQDKGIKVIOLTDLVAETYDLASQEAKDKLIEEFLEDSEPVLSFEHKVVVRNF LKAKKTSRELVEIMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFASAGNGISL N MKYVTRKRETIFAEFIFATHPDYKTTPH FDRLDEGNIEGGDVFIY KDTLVI
C4DS2
GVSERTNKEAILTIAKKlK KEAKF IVAINVPPMPNLMHLDT LTMVDKDKF LYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESI IGVKPVLIPI.1AGKGATQL DIDIETKFDGTNYLTIAPGVVVGYSRNIKTEAALRAAGVTVLSFEGNQLSLGMGS ARCMSMPLVREDVK
MSKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 30 K LQDKGIKVIQLVDLIVETYDLASKEAKEKLLEEFLDDSVPVLSDEKRATVKKF LQSQKS RSLVEYMIAGI KHDLKIESDLELIVDPMPNLYFTRDPFASAGNGISL MKYVTRKRE IFAEFIFATHPDYKTTPHWFDRLDEG IEGGDVFIYNKDTLVI
C4DS3
GVSERT KEAIL IAKKIKN KEAKFKKIVAINVPPMPNLMHLDT LTMVDKDKF LYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESIIGVKPVLIPIAGKGATQL D DIETHFDGTNYL IAPGVVVGYSRNIKTEAALRAAGVTVLSFEG QLSLGMGS ARCMSMPLVREDVK MSKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 31 KILQDKGIKVIQLTDLVTETYDLASQEAKDNLIEEFLEDSEPVLTEELKSWRTY LKSIKSTRELIQMMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFASAGNGISL NNMKYVTRKRETIFAEFIFATHPDYKTTPHWFDRLDEG IEGGDVFIYNKDTLVI
C4DS5
GVSERTN EAILTIAKKTK NKEAKFKKIVAINVPPMPNLMHLDT LTMVDKDKF LYSP LSVLKV EIDLSKEIE VETNKPLADVLESI IGVKPVLIPIAGKGATQL DIDIETHFDGTNYL IAPGVWGYSRNIKTEAALRAAGVTVLSFEGNQLSLGMGS ARCMSMPLVREDVK
MSKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 32 KILQDKG KVIQLTDLVAETFDLASKEEQEKL EEFLEDSEPVLSEAHKTAVRKF
ijTSRKSTREMVEFMMAGITKYDLGIEADPIELIVDPMPNL DPFASAGNGISL
C DS6 NNMKYVTRKRE IFAEFIFATHPDYKTTPHWFDRLDEG IEGGDVFIY KDTLVI
GVSER NKEAILT AKKIKNNKEAKFKKIVAINVPPMPNLMHLDTWLTMVDKDKF LYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESI IGVKPVLIPIAGKGATQL D DIETHFDGT YLTIAPGVWGY'SR IKTEAALRAAGVTVLSFEGNQLSLGMGS ARCMS PLVREDVK
MSKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 33 KILQDKGIKVIQLTDLVSETYDMVSKEKQEKLIEEFLEDSEPVLSEEHKGLVRKF LKSLKSSKELIQYM AGITKHDLNIEADHELIVDPMPNLYFTRDPFASAGNGISL MKYVTR RETIFAEFIFATHPDYKTTPHWFDRLDEG IEGGDVFIYNKDTLVI
C4DS7
GVSERTNKEAILTIAKKTKNNKEAKFKKIVAINVPPMPNLMHLDTWLTMVDKDKF LYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESI IGVKPVLIPIAGKGATQL DIDIETHFDGTNYLTIAPGVVVGYSRNIKTEAALRAAGVTVLSFEGNQLSLGMGS ARCMSMPLVREDVK
MSKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 34 KILQDKGIKVIQLSDLVAETYVKYATAEQKAAFIEKYLDEATPALSAENRERAKK YILSLEMQPVKMIRTMMAGLSKYELNVESNIELIIDPMPNLYFTRDPFASAGNGI SLNNMKYVTRKRETIFAEFIFATHPDYKTTPHWFDRLDEGNIEGGDVFIYNKDTL
C DS8
VIGVSERTNKEAIL IAKKIKNNKEAKFKKTVAINVPPMPNLMHLDTWLTMVDKD KFLYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESI IGVKPVLIPIAGKGAT QLDIDIETHFDGTNYLTIAPGWVGYSRNIKTEAALRAAGVTVLSFEGNQLSLGM GSARCMSMPLVREDVK
MSKINVYSEIGELKEVLVHTPGDEIRRISPSRLDELLFSAILEPNEAIKEHKGFL 35 KILQDKGIKVIQLSDLVAETYKHYASEAEKEAFIEKYLDEATPVLSKDMRAKVKN YILSMQGEPVKMVRTM AGVSKQELNVESEVELIVDPMPNLYFTRDPFASAGNGI SLNNMKYVTRKRET FAEFIFATHPDYKTTPHWFDRLDEG IEGGDVFIYNKDTL
C4DS9
VTGVSERTNKEAILTIAKKIKNNKFAKFKKIVATNVPPMPNLMHT-DT LTMVDKD KFLYSPNMLSVLKVWEIDLSKEIEMVETNKPLADVLESI IGVKPVLIPIAGKGAT QLDIDIETHFDGTNYLTIAPGWVGYSRNIKTEAALRAAGVTVLSFEGNQLSLGM GSARCMSMPLVREDVK
MSVFDSKFNGIHVYSEIGELESVLVH.EPGREIDYITPARLDELLFSAILESHDAR 36 KEHKLFVSELKANDINWELDELVAQTYDQVDQKIKDEFIDQWLQEAKPVLNDQL KKLVKNYLLKSQKEFSTKKMVRIMMAGIDKKEINIDLDRDLVVDPMPNLYFTRDP FASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTPWYYDPSLKLSIEGGDV
C5DS1 FIYNNNTLVVGVSERTDLETVTLLAKNIVANKECEFKRTVAINVPKWTNLMHLDT
WLTMLDKDKFLYSPIANDVFKFWDYDLVNGGEEPQPVENGLPLEGLLESl i.NKK.P ILIPIAGEGASQIDIERETHFDGTNYLAIRPGVVIGYSRNEKTNAALEAAGIKVL PFHGNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 37
KEHKLFVSELKANDIN ELIDLVAETYDLASQEAKDKLIEEFLEDSEPVLSEEH KVVVRNFLKAKKTSRELVEIMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTPWYYDPSLKLSIEGGDVFIY
C5DS2
NNNTLWGVSERTDLETVTLLAKNIVANKECEFKRIVAINVPKWTNLMHLDTWLT MLDKDKFLYSPIANDVi''Kb1 DYDLVNGGEEPQPVENGLPLEGLLESi INKKPILi P AGEGASQ DIERETHFDGTNYLAIRPGVVIGYSRNEK NAALEAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 38 KEHKLFVSELKANDTNVVELVDLIVETYDLASKEAKF.KLLEEFLDDSVPVLSDEH
C5DS3
RATVKKFLQSQKSTRSLVEYMIAGITKHDLKIESDLELIVDPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTPWYYDPSLKLSIEGGDVFIY NNTLWGVSERTDLETVTLLAKNIVANKECEFKRIVATNVPKWTNLMHLDTWLT
MLDKDKFLYSPIA DVFKFWDYDLV GGEEPQPVENGLPLΞGLLES11MKKPILI PIAGEGASQIDTERETHFDGTNYLAIRPGVVIGYSRNEKTNAALEAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGTHVYSEIGELESVIA/HEPGREIDYITPARLDELLFSAILESHDAR 39 KEHKLFVSELKANDIN ELSDLVAETYTYI-IATQKEREAFIEKWLDEAEPALTKD LRAKVKSyVLSKEGTPVAMVRTMMAGVSKQELNVESETELWDPMPNLYFTRDPF ASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTPWYYDPSLKLSIEGGDVF
C5DS4
TYNNNTLWGVSERTDLE VTLLAKNTVA KECEFKRIVAINVPKWTNLMHLDTW LTMLD DKFLYSPlANDVFKFWDYDLV GGEEPQPVENGLPLEGLLESIINKKPl LIPIAGEGASQIDIERETHFDGTNYLAIRPGWIGYSR EKTNAALEAAGIKVLP FHGNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKF GIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 40 KEHKLFVSELKANDI WELTDLVAETFDLASKEEQEKLIEEFLΞDSΞPVLSEAH KTAVRKFLTSRKSTREMVEFMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS
C5DS6 VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTP YYDPSLKLSIEGGDVFIY
NNTLWGVSERTDLETVTLLAKNIVANKECEFKRIVATNVPKWTNLMHLDTWLT MLDKDKfc'LYSPIANDVFKF DYDLV GGEEPQPVENGLPLEGLLESIINKKPlLl PIAGEGASQIDIERETHFDGTNYLAIRPGWIGYSRNEKTNAALEAAGI VLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKF GIHVYSEIGELESVLVHEPGREIDYITPARLDELLFSAILESHDAR 1 KEHKLFVSELKA DINWEL DLVSE YDMVSKEKQΞKLIEEFLEDSEPVLSEEH KGLVRKFLKSLKSSKELIQYMMAGITKHDLNIEADHELIVDPMPNLYFTRDPFAS
C5DS7 VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVNTP YYDPSLKLSIEGGDVFIY
KNTLWGVSERTDLETVTLLAK IVANKECEFKRIVAI VPK TNLMHLDTWLT MLDKDKFLYSPIAND b'KFWDYDLVNGGEEPQPVENGLPLΞGLLESi INKKPILi PI.AGEGASQIDIERETHFDGTNYLAIRPGWIGYSRNEKTNAALEAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVK
MSVFDSKFNGIHVYSEIGELETVLVHEPGREIDYITPAP.LDELLFSAILESHDAR 42 KEKQSFVKQLKDNGINWELDELVAQTYDQVDQKIKDEFIDQWLQEAKPVLNDQL KKLVKNYLLKSQKEFSTKKMVRIMMAGIDKKEINIDLDRDLWDPMPNLYFTRDP FAS GNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVKTPWYYDPAMKMSIEGGDV
C6DS1
FIYNNDTIWGVSERTDLETITLLAKNTKANKEVEFKRIVAINVPKWTNLMHLDT LTMbDKDKFLYSPIANDVF FiiDYDLVNGGAEPQPKENGLPLEGbLQSiiNKKP VLIPIAGNNASHIDIERETHFDGTNYLAIKPG VIGYARNEKTNAALAAAGIKVL PFHGNQLSLGMGMARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELETVLVHEPGREIDYITPARLDELLFSAILESHDAR 43
KEHQSFVKQLKDNGINWELIDLVAETYDLASQEAKDKLIEEFLEDSEPVLSEEH KVVVRNFLKAKKTSRELVEIMMAGITKYDLGIEADHELIVDPMP LYFTRDPFAS VGNGVTIHYMRYKVRORETLFSRFVFSNHPKLVKTPWYYDPAMKMSIEGGDVFIY
C6DS2
DTLWGVSERTDLETITLLAK IKANKEVEFKRIVAI VPK TNLMHLDTWLT MLDKDKF LYSPIAND i''Kb1WDYDLV GGAEPQPKENGLPLEGLLQSi INKKP Li PIAGNNASHIDIERETHFDGTNYLAIKPGVVIGYARNEKTNAALAAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELETVLVHEPGREIDYITPARLDELLFSAILESHDAR 44 KEKQSFVKQLKDNGTNVVELVDLIVETYDLASKEAKEKLLEEFLDDSVPVLSDEH RAT KKFLQSQKSTRSLVEYMIAGITKHDLKIESDLELI DPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVKTPWYYDPAMKMSIEGGDVFIY
C6DS3
NDTLWGVSERTDLETITLLAKNI ANKEVEF RIVAI VPK TNLMHLDT LT MLDKDKi'LYSPIANDVJ;'KFvJDYDLVNGGAEPQPKENGbPLEGLLQSIINKKPV LI PIAGNNASHIDIERETHFDGTNYLAIKPGVVIGYARNEKTNAALAAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELETVLVHEPGREIDYITPARLDELLFSAILESHDAR 45 KEHQSFVKQLKDNGINVVELSDLVAETYTYHATQKEREAFIEKWLDEAEPALTKD LRAKVKSYVLSKEGTPVAMVRTMMAGVSKQELNVESETELVVDPMPNLYFTRDPF
C6DS4 ASVGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVKTPWYYDPAMKMSIEGGDVF
IYNNDTLWGVSERTDLETI LLAKNIKANKEVEFKRIVAINVPK TNLMI-ILDT LTMLDKDKFLYSPIANDVFKF'DYDLVNGGAEPQPKENGLPLEGLLQSI INKKPV LIPIAGNNASKIDIERETHFDGTNYLAIKPGVVTGYARNEKTNAALAAAGIKVLP FHGNQLSLGMGNARCMSMPLSRKDVKW SVFDSKF GIHVYSEIGELETVLVHEPGREIDYITPARLDELLFSAILESHDAR 46 KEHQSFVKQLKDNGINWELTDLVTETYDLASQEAKDNLIEEFLEDSEPVLTEEL KSVVRTYLKSTKSTRELIQMMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVKTP YYDPAMKMSIEGGDVFIY
C6DS5
NNDTLVVi~VSERTDLET LLAKNIKANKEVEFKRIVAINVPKWTNLMHLDTWLT MLDKDKFLYSPIANDVFKFWDYDLVNGGAEPQPKENGLPLEGLLQS I INKKPVLI PIAG NASHIDIERETHFDGT YLAIKPGVVIGYARNEKTNAALAAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELETVLVHEPGREIDYITPARLDELLFSAILESHDAR 47 KEKQSFVKQLKDNGI VVELTDLVSETYDMVSKE.KQEKLTEEFLEDSEPVLSEEH KGLVRKFLKSLKSSKELIQYMMAGITKKDLNIEADKELIVDPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFVFSNHPKLVKTP YYDPAMKMSIEGGDVFIY
C6DS7
DTLWGVSERTDLETITLLAKNIKAN EVEFKRIVATNVPKWTNLMHLDTWLT MLDKDKFLYSPIANDVFKFWDYDLVNGGAEPQPKENGLPLEGLLQS I INKKPVLI P AGNNASH D ERETHFDGTNYLAIKPGWIGYARNEKTNAALAAAGIKVLPFH GNQLSLGMGNARCMSMPLSRKDVKW SVFDSKFNGIHVYSEIGELQTVLVHEPGREIEYITPARLDELLFSAILESHDAR. 48 KEHQEFVAELKKN INVVELDELVAQTYDQVDQK KDEFIDQWLQEAKPVLNDQL KLVKKYLLKSQKEFST KMVRIMMAGIDKKEINIDLDRDLWDPMPNLYFTRDP
c,DS 1 FASVGNGVTIHYMRYKVRQRETLFSRFIFANKPKLMNTPLYYNPDMKLSIEGGDV
FVYNNETLVVGVSERTDLDTITLLAKNIKANKEREFKRIVATNVPKWTNLMHLDT WLTMLDKDKFLYS IANDVFKFWDYDLVNGGDF.PQPKVNGLPLEKLLES T TNKKP ILIPIAGTSASNIDVERETKFDGTNYLAIAPGVVIGYSRNVKTNEALEAAGIKVL PFKGNQLSLGMGNARCMSMPLSRKDVKW SVFDSKFNGIHVYSEIGELQTVLVH.EPGRE EYITPARLDELLFSA LESHDAR 49 KEHQEFVAELKK NI WELIDLVAETYDLASQEAKDKLIEEFLEDSEPVLSEEH KVVVR FLKAKKTSRELVEI MAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFIFANHPKLMNTPLYYNPDMKLSIEGGDVFVY
C7DS2
NNETLVVGVSERTDLDTI LLAKNIKANKEREFKRIVATNVPKWTNLMHLDTWLT MLDKDKfc'LYSPIA DVFKF DYDLV GGDEPOPKV GLPLEKLLESIINKKPlLl PIAGTSASNIDVERETHFDGTNYLAIAPGVVIGYSRNVKTNEALEAAGIKVLPFK. GNQLSLGMGNARCMSMPLSRKDVKW SVFDSKFNGIHVYSEIGELQTVLVHEPGREIEY TPARLDELLFSAILESHDAR 50 KEHOEFVAELKKNNIN ELVDLIVETYDLASKEAKEKLLEEFLDDSVPVLSDEH PATVKKFLQSQKSTRSLVEYMIAGITKHDLKIESDLELIVDPMPNLYFTRDPFAS VGNGVTIHYMRYKVRQRETLFSRFIFANHPKLMNTPLYYNPDMKLSIEGGDVFVY
C7DS3
NNETL GVSERTDLDTI LLAKNIKANKEREFKRIVAINVPKWTNLMHLDT LT MLDKDKFLYSPIANDVb' .b1 DYDLV GGDEPQPK.V GLPLEKLLES J. KKPILi PIAGTSASNIDVERETHFDGTNYLAIAPGWIGYSRNVKTNEALEAAGIKVLPFK GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELQTVLVH.EPGRE EYITPARLDELLFSA LESHDAR 51 KEHQEFVAELKKNNINWELSDLVAETYTYHATQKEREAFIEKWLDEAEPALTKD LRAKVKSYVLSKEGTPVAMVRTMMAGVSKQELNVESETELVVDPMPNLYFTRDPF ASVGNGVTIHYMRYKVRQRETLFSRFIFANHPKLMNTPLYYNPDMKLSIEGGDVF
C7DS4
VYNNETLVVGVSP.RTDLDTITLLAK IKANKEREFKRTVAI VPKWTNLMHLD W LTMLDKDKFLYSPIANDVFKFWDYDLVNGGDEPQPKVNGLPLEKLLESIiNKKPI LIPIAGTSASNIDVERETHFDGTNYLAIAPGWIGYSRNVKTNEALEAAGIKVLP FKGMQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELQTVLVHEPGREIEYITPARLDELLFSAILESHDAR 52 KEHQEFVAELKKNNIN ELTDLVTETYDLASQEAKDNLIEEFLEDSEPVLTEEL KSVVRTYLKSIKSTRELIQMMMAGITKYDLGIEADHELIVDPMPNLYFTRDPFAS VGNGV IHYMRYKVRQRETLFSRFIFANHPKLMN PLY NPDMKLSIEGGDVFVY
C7DS5
ETLWGVSERTDLDTITLLAK IKANKEREFKRIVAI VPK TNLMHLDTWLT MLDKDKFLYSPIANDVi<'Kb1 DYDLVNGGDEPQPKVNGLPLEKLLES i INKKPILi PIAGTSASNIDVERETHFDGTNYLAIAPGWIGYSRNVKTNEALEAAGIKVLPFK GNQLSLGMGNARCMSMPLSRKDVKW
MSVFDSKFNGIHVYSEIGELQTVLVHEPGREIEYITPARLDELLFSAILESHDAR 53 KEHQEFVAELKKNNINWELTDLVAETFDLASKEEQEKLIEEFLEDSEPVLSEAH
C7DS6
KTAVRKFLTSRKSTREMVEFMMAGITKYDLGIEADKELIVDPMPNLYFTRDPB'AS VGNGVTIHYMRYKVRQRETLFSRFIFANHPKLMNTPLYYNPDMKLSIEGGDVFVY NETLWGVSERTDLDTITLLAKNIKANKEREFKRIVATNVPKWTNLMHLDTWLT
MLDKDKFLYSPIA DVFKPWDYDLVNGGDEPQPKVNGLPLEKLLP.STINKKPILT PIAGTSASNIDVERETHFDGTNYLAIAPGWIGYSR VKTNEALEAAGIKVLPFK
GNQLSLGMGNARCMSMPLSRKDVKW
MSKIRVYSEIG LKKYLVHTPGDEIRRISPSRT-FFiLLFSAVLEPNAAIEEHKRFV 54 KLLEDRGIQAIOijVDLIVETYDLAS EAKEKLLEEFJjDDSVPVLSDEHRA VKKF LQSQKSTRSLVEYMIAGITKHDLKIESDLELIVDPMPNLYFTRDPFASAGNGISL MKYWR RETIFAEFIFAIHPEY ETPHWFDRLDHGSIEGGDVFVYNKDTLVI
C8DS3
GVSERTNKEAIITIAKHTQDNKEARFKKIVAI VPP PNLMHLDTWLT VDKNKF IYSPNMLS LKIWEIDLAKPIEMVESNKSLTEVLES 11GEKPILI PIAGEGASQL DIDIETHFDG YLTIAPGVWGYSRNEKTEKALKAAGITVLSFEGNQLSLGMGS ARCMSMPLVREDVK
MSKIRVYSEIG LKKVLVHTPGDEIRRISPSRLEELLFSAVLEPNAAIEEHKRFV 55 TJ-P-DRGIOAIOLSDLVAETYTYHATOKEREAFIEKWLDEAEPALTKDLRAKVKS
YVLS EGTPVAMVRTMMAGVSKQEL VESETELWDPMPNLYFTRDPFASAGNGI SLNNMKYVVRKRETIFAEFIB'AIHPEYKETPHWFDRLDHGSIEGGDVFVYNKDTL
C8DS4
VIGVSERTNKEAITTIAKHIQDNKEAEFKKTVAI VPPMPNLMHLDTWLTMVDKN KFIYSP MLSVLKIWEIDLAKPIEMVESNKSLTEVLESIIGE PILIPIAGEGAS QLDIDIETHFDGTNYLTIAPGVWGYSRNEKTE ALKAAGITVLSFEGNQLSLGM GSARCMSMPLVREDVK
MSKIRVYSEIG LKKYLVHTPGDEIRRISPSRT.FRLLFSAVLEPNAAIEEHKRFV 56 TJ.RDRGIQAIOLSDLVAETYKHYASEAEKEAFIEKYLDEATPVLSKDMRAKVK YILSMQGEPVKMVRTM AGVSKQEL VESEVELIVDPMPNLYFTRDPFASAGNGI SLNNMKYWRKRETIFAEFIFAIHPEYKETPHWFDRLDHGSIEGGDVFVYNKDTL
C8DS9
VIGVSERTNKEAIITIAKHIODNKEAEFKKIVAI VPPMPNLMHT.DTWLTMVDK KF IYS PNMLSVLKIWE IDLAKPIEMVESNKSLTEVLESIIGEKPILI PIAGEGAS QLDIDIETHFDG NYLTIAPGVVVGYSR EKTEKALKA GITVLSFEG QLSLG GSARCMSMPLVREDVK
MSKI VYSEIGVL EVLVHTPGDEIRRIAPSRLDELLFSAILEPSAAIQEHKSFL 57 KILQDRGIKTIOLVDLIVETYDLASKEAKEKLLEEFLDDSVPVLSDEHRATV KF LQSQKSTRSLVEYMIAGITKHDLKIESDLELIVDPMPNLYFTRDPFASAGNGISL
NNMKYVVRKRETIFAEFIFSIHPEYKKTPHWFDRLDNGSIEGGDVFIYNKDTLVI
C9DS3
GVSERTNKEAIITIAKHIQDNKEAQF KIVAINVPPMPNLMHLDTWLTMVDK KF LYSPNMLSVLKVWEIDLSKPIEMVETNKPLAEVLESIIGEKPILIPIAGKDATQL DIDIETKFDGTNYLTIAPGVVVGYSRNVKTEAALRAAGVTVLSFEGNQLSLGMGS ARCMS PLVREDVK
MSKI VYSEIGVLKEVLVHTPGDEIRRIAPSRLDELLFSAILEPSAAIQEHKSFL 58 KI LQDRGIK IQLSDL AETYTYHATQKEREAFIEK LDEAEPALTKDLRAKVKS YVLSKEGTPVAMVRTMMAGVSKQELNVESETELWDPMPNLYFTRDPFASAGNGI SLNN KY VRKRET I FAEFI FS IHPEYKKTPHWFDRLDNGS IEGGDVFIYNKDTL
C9DS4
VIGVSERTNKEAIITIAKHIQDNKEAQFKKIVAI VPPMPNLMHLDTWLTMVDK KFLYSP MLSVLKVWEIDLSKPIEMVET KPLAEVLESI IGEKPILIPIAGKDAT QLDIDIETHFDGTNYLTIAPGV GYSR VKTEAALRAAGVTVLSFEGNQLSLGM GSARCMSMPLVREDVK
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C9DS8
vigvsertnkea i i tiakhiqdnkeaqf kki vainvpprapnlmh Ldtwltmvdkn kfIyspnm1 sv1 kv eid1s k.pί ernvetri kp1aev1es i igekpil i piagkda t; qA.didiethfdgtny11±apgvv gys rn v teaa1 raagvtvlsfegnqislgm gsarcmsmp1vredvk
Hence, in some embodiments, the chimeric ADI comprises, consists, or consists essentially of an iilustrative chimeric sequence from Table A2 (SEQ ID NOs:4-13 or 22-59), or a variant or fragment thereof having ADI activity. Certain embodiments include variants of the reference ADI polypeptide sequences described herein, whether described by name or by reference to a sequence identifier (e.g., Tables A1-A2). A 'Variant" sequence, as the term is used herein, refers to a polypeptide or polynucleotide sequence that differs from a reference sequence disclosed herein by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Certain variants thus include fragments of a reference sequence described herein. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.
in many instances, a biologically active variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When if is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since if is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.
in making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittie, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doo!ittie, 1982). These values are: isoieucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1 .9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (■■■ 3.2); giutamate (-3.5); g!utamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U. S. Patent 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1 ); giutamate (+3.0 ± 1 ); serine (+0.3); asparagine (+0.2); g!utamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1 ); alanine (-0.5); histidine (■■■ 0.5); cysteine (-1.0); methionine (-1 .3); valine (-1.5); leucine (-1.8); isoieucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). it is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; giutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoieucine.
Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophi!icity values include leucine, isoieucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1 ) aia, pro, giy, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, lie, leu, met, aia, phe; (4) iys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain non-conservative changes. In a preferred embodiment, variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.
In certain embodiments, a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids in length, including all integers in between, and which may comprise ail or a portion of a reference sequence (see, e.g., Sequence Listing).
in other specific embodiments, a polypeptide sequence consists of about or no more than about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids, including all integers in between, and which may comprise ail or a portion of a reference sequence (see. e.g., Sequence Listing).
in still other specific embodiments, a polypeptide sequence is about 10-1000,
10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10- 30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20- 100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50- 300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids, including ail ranges in between, and comprises ail or a portion of a reference sequence, in certain embodiments, the C-ierminai or N-termina! region of any reference polypeptide may be truncated by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400- 450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101 , 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide. Typically, the bio!ogica!iy-active fragment has no less than about 1 %, about 5%, about 10%, about 25%, or about 50% of an activity of the biologically-active reference polypeptide from which it is derived.
in general, variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identify or sequence homology to a reference polypeptide sequence. Moreover, sequences differing from the native or parent sequences by the addition [e.g., C- terminai addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges in between) but which retain the properties or activities of a parent or reference polypeptide sequence are contemplated.
in some embodiments, variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). in other embodiments, variant polypeptides differ from a reference sequence by at least 1 % but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.)
Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, in a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (J. Moi. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Biossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Biossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:1 1-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a
"query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLAST and X BLAST programs (version 2.0) of Altschul, et a/., (1990, J. Moi. Bioi, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordiength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., {Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and N BLAST) can be used.
in one embodiment, as noted above, polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool. A local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared. A modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs). The results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.
The raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).
Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty. For a gap of length n, the gap cost would be G+Ln. The choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 1 1 , and a low value for L (1-2) e.g., 1 .
The bit score, S!, is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms "bit score" and "similarity score" are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.
The E-Value, or expected value, describes the likelihood that a sequence with a similar score will occur in the database by chance, it is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e"11 ? means that a sequence with a similar score is very unlikely to occur simply by chance. Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related.
Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 1 1 and a gap extension penalty of 1
In one embodiment, sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 1 1 and a gap extension penalty of 1.
in a particular embodiment, sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Acceirys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA.
89:10915-10919, 1992). GAP uses the algorithm of Needleman and Wunsch ( J Mo! Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
in one particular embodiment, the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 280, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more, including all integers and ranges in between, wherein the BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 1 1 , and a gap extension penalty of 1.
As noted above, a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et a!., (Methods in Enzymo!. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et a!., ("Molecular Biology of the Gene," Fourth Edition, Benjamin/Cummings, Menio Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et a!., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).
Methods for screening gene products of combinatorial libraries made by such modifications, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of reference polypeptides. As one example, recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89: 781 1-7815, 1992; Deigrave et ai, Protein Engineering. 6: 327-331 , 1993).
Native ADI may be found in microorganisms and is immunogenic and rapidly cleared from circulation in a patient. These problems may be overcome by engineering ADi to reduce its antigenicity, such as by engineering chimeric ADi molecules. In one
embodiment, chimeric ADi are constructed by combining different domains (e.g. catalytic domain and a-heiicai domains) from different ADi proteins using standard molecular biological, or protein synthesis techniques. In one embodiment, the catalytic domain from M. arginini or M. arthritidis is combined with the a-heiical domain of M. hominis. In another embodiment, the catalytic domain of M. arginini is combined with the a-helical domain of M. arthritidis. in a further embodiment, the catalytic domain of M. arthritidis is combined with the a-helicai domain of M. arginini. As would be recognized by the skilled person, other combinations of catalytic and a-helical domains can be constructed from ADI proteins derived from other species, such as from Mycoplasma pneumoniae, Steptococcus pyogenes, Steptococcus pneumoniae, Borreiia burgdorferi, Borreiia afzeiii, Giardia intestinaiis, Clostridium perfringens, Bacillus iicheniformis, Enterococcus faecaiis, and Lactobacillus sake.
Antigenicity problems may also be overcome by modifying chimeric ADI. Thus, the present disclosure provides chimeric ADi modified by a modifying agent, including, but not limited to macromolecule polymers, proteins, peptides, polysaccharides, or other compounds. Arginine deiminase or chimeras thereof as described herein and the modifying agent may be linked by either covalent bonds or non-covalent interaction to form a stable conjugate or a stable composition to achieve a desired effect, in certain embodiments, the modified chimeric ADI retains the biological activity of an unmodified chimeric ADI and has a longer half-life in vivo and lower antigenicity than the unmodified, chimeric ADI. In certain embodiments, the modified chimeric ADI retains at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 80%, 85%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the biological activity of unmodified chimeric ADi. in one embodiment, a modifying agent can be a polymer or a protein or a fragment thereof that is biocompatible and can increase the half-life of chimeric ADi in blood. The modifying agent can be either chemically coupled to chimeric ADi or where applicable, linked to the chimeric ADI via fusion protein expression.
Macromo!ecuie polymers may include a non-peptide macromolecu!e polymer, which in certain embodiments, may have its own bioactivity. Suitable polymers include, but are not limited to, polyenoi compounds, poiyether compounds, polyvinylpyrrolidone, poly amino acids, copolymer of divinyi ether and maleic anhydride, N-(2-hydroxypropy1)~ methacrylamide, polysaccharide, polyoxyethylated po!yo!, heparin or its fragment, poly-aikyi- ethylene glycol and its derivatives, copolymers of poly-alkyl-ethylene glycol and its derivatives, polyvinyl ethyl ether), a,P-Po!y[(2-hydroxyethy!)-DL-aspartamide].
polycarboxylates, poly oxyethyiene-oxymethy!enes, poiyacryloyl morpholines, copolymer of amino compounds and oxyolefin, poly hyaluronic acid, polyoxiranes, copolymer of ethanedioic acid and maionic acid, poly (1 ,3-dioxoiane), ethylene and maleic hydrazide copolymer, poly sialic acid, cyclodextrin, etc. in certain embodiments, the polymer is polyethylene glycol.
The polyenoi compounds as used herein include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxyi polyethylene glycol), polyvinyl alcohol, polyailyi alcohol, polybuteno! and the like, and their derivatives, such as lipids.
The poiyether compounds include, but are not limited to poly alkylene glycol (HO((CH2)xO)nH), polypropylene glycol, polyoxyrehylene (HO((CH2)20)nH), polyvinyl alcohol ((CH2CHOH)n).
Poly amino acids include, but are not limited to, polymers of one type of amino acid or copolymers of two or more types of amino acids, for example, polyalanine or polylysine, or block co-polymers thereof.
Polysaccharides include but are not limited to, glucosan and its derivatives, for example dextran sulfate, cellulose and its derivatives (including methyl cellulose and carboxymethyi cellulose), starch and its derivatives, polysucrose, etc.
In one specific embodiment of the present invention, chimeric ADI is modified by coupling with proteins or peptides, wherein one or more proteins or peptides are directly or indirectly linked to chimeric ADi. The proteins can either be naturally existing proteins or their fragments, including but not limited to naturally existing human serum proteins or their fragments, such as thyroxine-binding protein, transthyretin, a1-acid glycoprotein, transferrin, fibrinogen, immunoglobulin, ig Fc regions, albumin, and fragments thereof. By "fragment" is meant any portion of a protein that is smaller than the whole protein but which retains the desired function of the protein. Engineered chimeric ADl may be directly or indirectly linked to a protein via a covalent bond. Direct linking means that one amino acid of chimeric ADl is directly linked to one amino acid of the modifying protein, via a peptide bond or a disulfide bridge, indirect linking refers to the linkages between a chimeric AD! and a modifying protein, via originally existing chemical groups there between or specific chemical groups added through biological or chemical means, or the combination of the above-mentioned linkages.
In one particular embodiment, chimeric ADl is modified by covalent attachment with PEG. Chimeric ADl covalentiy modified with PEG (with or without a linker) may be hereinafter referred to as chimeric "ADi-PEG." When compared to unmodified chimeric ADl, chimeric ADi-PEG retains most of its enzymatic activity, is far less antigenic, has a greatly extended circulating half-life, and is much more efficacious in the treatment of tumors.
"Polyethylene glycol" or "PEG" refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH2CH2)nOH, wherein n is at least 4. "Polyethylene glycol" or "PEG" is used in combination with a numeric suffix to indicate the approximate weight average molecular weight thereof. For example, PEGS, 000 refers to PEG having a total weight average molecular weight of about 5,000; PEG12,000 refers to PEG having a total weight average molecular weight of about 12,000; and PEG20,000 refers to PEG having a total weight average molecular weight of about 20,000.
in one embodiment of the present invention, the PEG has a total weight average molecular weight of about 1 ,000 to about 50,000; in one embodiment from about 3,000 to about 40,000, and in another embodiment from about 5,000 to about 30,000; in certain embodiments from about 8,000 to about 30,000; in other embodiments from about 1 1 ,000 to about 30,000; in additional embodiments, from about 12,000 to about 28,000; in still other embodiments, from about 16,000 to about 24,000; and in other embodiments, about 18,000 to about 22,000; in another embodiment, from 19,000 to about 21 ,000, and in one embodiment, the PEG has a total weight average molecular weight of about 20,000. Generally, PEG with a molecular weight of 30,000 or more is difficult to dissolve, and yields of the formulated product may be reduced. The PEG may be a branched or straight chain. Generally, increasing the molecular weight of the PEG decreases the antigenicity of the ADl or chimeric ADl. The PEG having a molecular weight described in this embodiment may be used in conjunction with chimeric ADl, and, optionally, a biocompatible linker, to treat cancer, including, for example, acute myeloid leukemia, such as relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, stomach cancer and esophageal cancer.
In another embodiment of the present invention, the PEG has a total weight average molecular weight of about 1 ,000 to about 50,000; in certain embodiments about 3,000 to about 30,000; in other embodiments from about 3,000 to about 20,000; in one embodiment from about 4,000 to about 12,000; in still other embodiments from about 4,000 to about 10,000; in additional embodiments from about 4,000 to about 8,000; still further embodiments from about 4,000 to about 6,000; and about 5,000 in another embodiment. The PEG may be a branched or straight chain, and in certain embodiments is a straight chain. The PEG having a molecular weight described in this embodiment may be used in conjunction with chimeric AD!, and optionally, a biocompatible linker, to treat graft versus host disease (GVHD) or cancer.
While chimeric ADI-PEG is the illustrative modified chimeric AD! described herein, as would be recognized by the skilled person chimeric AD! may be modified with other polymers or appropriate molecules for the desired effect, in particular reducing antigenicity and increasing serum half-life.
Chimeric ADI may be covalently bonded to a modifying agent, such as PEG, with or without a linker, although a preferred embodiment utilizes a linker.
The linker used to covalently attach chimeric ADI to a modifying agent, e.g.
PEG, may be any biocompatible linker. As discussed above, "biocompatible" indicates that the compound or group is non-toxic and may be utilized in vitro or in vivo without causing injury, sickness, disease, or death. A modifying agent, such as PEG, can be bonded to the linker, for example, via an ether bond, a thiol bond, or an amide bond. The linker group includes, for example, a succinyi group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, and combinations thereof, in one embodiment, the source of the biocompatible linker is succinimidyl succinate (SS). Other suitable sources of linker may include an oxycarbonylimidazole group (including, for example, carbonylimidazole (GDI)), a nitro phenyl group (including, for example, nitrophenyi carbonate (NCP) or trichiorophenyl carbonate (TCP)), a trysylate group, an aldehyde group, an isocyanafe group, a vinylsuifone group, or a primary amine, in another embodiment, the linker is derived from SS, SPA, SCM, or NHS; in certain embodiments, SS, SPA, or NHS are used, and in other embodiments, SS or SPA are used. Thus, in certain embodiments, potential linkers can be formed from methoxy-PEG succinimidyl
succinate(SS), methoxy-PEG succinimidyl glutarate(SG), methoxy-PEG succinimidyl carbonate (SC), methoxy-PEG succinimidy! carboxymethyi ester (SCM), metboxy-PEG2 N- hydroxy succinimide (NHS), methoxy-PEG succinimidyl butanoate (SBA), methoxy-PEG succinimidy! propionate (SPA), methoxy-PEG succinimidyl glutaramide, and methoxy-PEG succinimidyl succinimide.
Alternatively, chimeric ADI may be coupled directly to a modifying agent, such as PEG (i.e., without a linker) through an amino group, a sulfhydryi group, a hydroxyl group or a carboxyl group.
Chimeric ADi may be covalently bonded to PEG, via a biocompatible linker, using methods known in the art, as described, for example, by Park et ai, Anticancer Res., 1 :373-376 (1981 ); and Zapiipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992), the disclosures of which are hereby incorporated by reference herein in their entirety.
The attachment of PEG to chimeric ADI increases the circulating half-life of chimeric ADi. Generally, PEG is attached to a primary amine of chimeric ADI. Selection of the attachment site of PEG, or other modifying agent, on the chimeric ADi is determined by the role of each of the sites within the active domain of the protein, as would be known to the skilled artisan. PEG may be attached to the primary amines of chimeric ADi without substantial loss of enzymatic activity. For example, ADi cloned from Mycoplasma arginini, Mycoplasma arthritidis and Mycoplasma hominis has a number of lysine residues that may be modified by this procedure. In other words, one or more or all of the lysines are possible points at which ADI and chimeric forms of ADi as described herein can be attached to PEG via a biocompatible linker, such as SS, SPA, SCM, SSA and/or NHS. PEG may also be attached to other sites on AD! and chimeric forms of ADI as described herein, as would be apparent to one skilled in the art in view of the present disclosure.
From 1 to about 30 PEG molecules may be covalently bonded to chimeric
ADI. In certain embodiments, chimeric ADI is modified with one PEG molecule. In other embodiments, chimeric ADI is modified with more than one PEG molecule. In one embodiment, chimeric ADi is modified with about 1 to about 10 PEG molecules, in one embodiment from about 2 to about 8 PEG molecules and in another embodiment, from about 9 to about 12 PEG molecules, in another embodiment, the chimeric ADI is modified with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 PEG molecules, in one specific embodiment, chimeric AD! is modified with 4.5 - 5.5 PEG molecules per ADi. in another embodiment, chimeric ADi is modified with 5 ± 1.5 PEG molecules.
in another embodiment, about 15% to about 70% of the primary amino groups in chimeric ADi are modified with PEG, in one embodiment about 20% to about 85%, about 25% to about 60%, or in certain embodiments about 30% to about 55%, or 45% to about 50%, and in other embodiments about 20% or 30% or 40% or 50% of the primary amino groups in arginine deiminase are modified with PEG. As would be understood by the skilled artisan, the range of primary amino groups depends upon how many lysines are successfully removed. In certain embodiments, all of the lysines may be removed and the N- terminus of the molecule is PEGyiated. When PEG is covalentiy bonded to the end terminus of chimeric ADI, it may be desirable to have only 1 PEG molecule utilized. Increasing the number of PEG units on chimeric ADI increases the circulating half-life of the enzyme.
However, increasing the number of PEG units on chimeric ADI decreases the specific activity of the enzyme. Thus, a balance needs to be achieved between the two, as would be apparent to one skilled in the art in view of the present disclosure.
in the present invention, a common feature of biocompatible linker is that they attach to a primary amine of arginine deiminase via a succinyl group. Once coupled with chimeric ADI, SS-PEG has an ester linkage next to the PEG, which may render this site sensitive to serum esterase, which may release PEG from chimeric ADI in the body. SPA- PEG and PEG2-NHS do not have an ester linkage, so they are not sensitive to serum esterase.
in certain embodiments, a biocompatible linker is used in the present invention. PEG which is attached to the protein may be either a straight chain, as with SS- PEG, SPA-PEG and SC-PEG, or a branched chain of PEG may be used, as with PEG2- NHS.
In certain embodiments, the chimeric ADI of the present disclosure may be modified as described in US Patent No. 6,835,462. In particular, modifications of one or more of the naturally occurring amino acid residues of ADI and chimeric molecules of ADI, in particular derived from Mycoplasma hominis, M, arthritidis and M. arginini, can provide for an enzyme that is more easily renatured and formulated thereby improving existing techniques for the manufacture of chimeric ADI and therapeutic compositions comprising the same. In one embodiment, the chimeric ADI of the present disclosure is modified to remove one or more lysine residues (e.g., the lysine can be substituted with another amino acid or analogues thereof, or a non-natural amino acid). In particular, in one embodiment, the chimeric ADI is modified to be free of the lysine at position 1 12, 374, 405 or 408 of SEQ ID NO:1 (M. hominis ADI), or a combination of one or more of these positions, in a further embodiment, the chimeric ADI is modified to be free of one or more lysines, for example, 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or more lysine residues, should they be present, can be substituted with another amino acid or analogues thereof, or a nonnatural amino acid. In one embodiment, a chimeric ADI has 5 lysines substituted, for example, at position 7, 88, 137, 209, and 380 of SEQ ID NO: 4. In another embodiment, a chimeric ADl has 10 lysines substituted, for example, at positions 7, 9, 59, 88, 1 15, 1 16, 137, 178, 209, and 380 of SEQ ID NO: 4, In yet another embodiment, a chimeric ADl has 15 lysines substituted, for example, at positions 7, 9, 59, 66, 88, 91 , 93, 1 15, 1 16, 137, 141 , 178, 209, 279, and at position 380 of SEQ ID NO: 4. In one embodiment, a chimeric ADl comprises 21 lysines substituted, for example, at positions 7, 9, 56, 59, 66, 88, 91 , 93, 96, 1 15, 1 16, 137, 141 , 178, 209, 254, 279, 325, 326, 380, and 406 of SEQ ID NO: 4,
Illustrative chimeric ADl molecules having lysine substitutions are set forth in SEQ ID NOs: 10-13.
in certain embodiments, pegylation sites associated with ADl located at or adjacent to the catalytic region of the enzyme are modified. For purposes of the present invention, the phrase "pegylation site" may be defined as any site or position of AD! or a chimeric ADl that may be covaiently modified with polyethylene glycol A "pegylation site" can be considered located at or adjacent to the catalytic region of the enzyme where pegylation of the site results in a significant reduction in catalytic activity of the enzyme. The pegylation of such sites has traditionally resulted in the inactivation of the enzyme. For example, ADl from Mycoplasma hominls has a lysine at the 1 12 position which can be considered to be at or adjacent the catalytic region of the enzyme. The attachment of PEG to this lysine at the 1 12 position can inactivate the enzyme, in addition, ADl from
Mycoplasma homlnis has a cysteine at the 397 position which can be considered to be at or adjacent the catalytic region of the enzyme. The amino acid substitutions for cysteine at the 397 position can inactivate the enzyme. In particular, substituting alanine, histidine, arginine, serine, lysine or tyrosine for cysteine at the 397 position can result in a loss of ail detectable enzyme activity. ADl from Mycoplasma homlnis also has three lysines located near this conserved cysteine, in particular Lys374, Lys405 and Lys408. The attachment of PEG to Lys374, Lys405, Lys408 or combinations thereof can inactivate the enzyme.
it is to be understood that ADl derived from other organisms may also have pegylation sites corresponding to 1 12 position of ADl from Mycoplasma hominls. For example, ADl from Steptococcus pyrogenes has lysine at the 104 position, ADl from
Mycoplasma pneumoniae has lysine at the 106 position, and ADl from Giardia intestinaiis has lysine at the 1 14 position. In addition, AD! from some organisms may have lysines corresponding to the same general location as the 1 12 position of ADl from Mycoplasma homlnis, The location of lysine in ADl from such organisms are known to the skilled person and are described in US Patent No. 6,635,462.
Thus, in one embodiment, the present invention provides for certain amino acid substitutions in the polypeptide chain of AD!. These amino acid substitutions provide for modified ADl that loses less activity when modified by a modifying agent, e.g., upon pegy!ation. By eliminating pegyiation sites, or other known modification sites, at or adjacent to the catalytic region of enzyme, optima! modification, e.g., pegyiation, can be achieved without the loss of activity.
It is to be understood that other embodiments of the invention are based on the understanding that certain structurai characteristics of arginine deiminase may prevent or interfere with the proper and rapid renaturation when produced via recombinant technology, in particular, these structural characteristics hinder or prevent the enzyme from assuming an active conformation during recombinant production. For purposes of the present invention, the phrase "active conformation" may be defined as a three-dimensional structure that allows for enzymatic activity by unmodified or modified arginine deiminase or chimeric arginine deiminase. The active conformation may, in particular, be necessary for catalyzing the conversion of arginine into citru!iine. The phrase "structural characteristic" may be defined as any trait, quality or property of the polypeptide chain resulting from a particular amino acid or combination of amino acids. For instance, arginine deiminase may contain an amino acid that results in a bend or kink in the normal peptide chain and thus hinders the enzyme from assuming an active conformation during renaturation of the enzyme. In particular, arginine deiminase from Mycoplasma hominis has a proline at the 210 position that may result in a bend or kink in the peptide chain, making it more difficult to renature the enzyme during recombinant production, it is to be understood that arginine deiminase derived from other organisms may also have sites corresponding to the 210 position of arginine deiminase from Mycoplasma hominis.
The present invention thus again provides for certain amino acid substitutions in the polypeptide chain of wild type arginine deiminases and chimeric arginine deiminases derived therefrom. Such amino acid substitutions can eliminate the problematic structurai characteristics in the peptide chain of arginine deiminase. Such amino acid substitutions provide for improved renaturation of the modified arginine deiminase. These amino acid substitutions make possible rapid renaturing of modified chimeric arginine deiminases using reduced amounts of buffer. These amino acid substitutions may also provide for increased yields of renatured modified chimeric arginine deiminase. in one embodiment of the invention, the modified chimeric arginine deiminase has an amino acid substitution at P210 or the equivalent residue. As mentioned above, arginine deiminase derived from
Mycoplasma hominis has the amino acid proline located at the 210 position. While not limiting the present invention, it is presently believed that the presence of the amino acid proline at position 210 results in a bend or kink in the normal polypeptide chain that increases the difficulty of renaturing (i.e., refolding) arginine deiminase. Substitutions for proline at position 210 make possible the rapid renaturation of modified arginine deiminase and chimeras derived therefrom using reduced amounts of buffer. Substitutions for proline at position 210 may also provide for increased yields of renatured modified chimeric arginine deiminase. In one embodiment, the proline at position 210 is substituted with serine. It is to be understood that in accordance with this aspect of the invention, other substitutions at position 210 may be made. Examples of other substitutions include Pro210 to Thr210.
Pro210 to Arg21 G, Pro210 to Asn21 G, Pro210 to Gln210 or Pro210 to Met210. By eliminating those structural characteristics associated with the amino acid of position 210 of the wild- type arginine deiminase and chimeras derived therefrom, proper refolding of the enzyme can be achieved.
The methods of the present invention can involve either in vitro or in vivo applications, in the case of in vitro applications, including ceil culture applications, the compounds described herein can be added to the cells in cultures and then incubated. The compounds of the present invention may also be used to facilitate the production of monoclonal and/or poiycional antibodies, using antibody production techniques well known in the art. The monocionai and/or poiycional antibodies can then be used in a wide variety of diagnostic applications, as would be apparent to one skilled in the art.
The in vivo means of administration of the compounds of the present invention will vary depending upon the intended application. Administration of the chimeric ADI compositions described herein, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions can be prepared by combining chimeric ADI, e.g., chimeric ADI-PEG, chimeric ADI-PEG 20, with an appropriate
physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other anti-cancer agents as described elsewhere herein) and/or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. Modes of administration depend upon the nature of the condition to be treated or prevented. Thus, chimeric ADI-PEG, e.g., chimeric ADI-PEG 20, may be administered orally, intranasaily, intraperitoneally,
parenteraliy, intravenously, intraiymphatical!y, intratumorly, intramuscularly, interstitia!iy, intra-arterially, subcutaneously, intraoculariy, intrasynoviai, transepithelial, and
transdermally. An amount that, following administration, reduces, inhibits, prevents or delays the progression and/or metastasis of a cancer is considered effective. In certain embodiment, the chimeric ADi compositions herein increase median survival time of patients by a statistically significant amount. In one embodiment, the chimeric ADI treatments described herein increase median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, chimeric ADi treatments increase median survival time of a patient by 1 year, 2 years, 3 years, or longer, in one embodiment, the chimeric ADi treatments described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer, in certain embodiments, the chimeric ADI treatments described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.
in certain embodiments, the amount administered is sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 50% decrease in tumor mass, or by altered {e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the amount administered is sufficient to result in stable disease. In other embodiments, the amount administered is sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.
in certain embodiments the amount administered is sufficient to inhibit NO synthesis, inhibit angiogenesis, and or is sufficient to induce apoptosis in tumor cells or any combination thereof. NO synthesis, angiogenesis and apoptosis may be measured using methods known in the art, see, e.g., Current Protocols in immunoiogy or Current Protocois in Molecular Biology, John Wiley & Sons, New York, N.Y.(2009 and updates thereto); Ausubel et a/., Short Protocols in Molecular Biology, 3"1 ed., Wiley & Sons, 1995; and other like references. In one particular embodiment the amount administered inhibits NO synthesis and inhibits the growth of melanoma and synergizes with other chemotherapies as described herein, such as cispiatin. Accordingly, one embodiment of the present disclosure provides a method of treating melanoma by administering chimeric ADI-PEG 20 in combination with cispiatin, wherein the treatment depletes endogenous nitric oxide (NO).
The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.
The chimeric ADI compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy,
transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics.
Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavaiiable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described chimeric ADI composition in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a chimeric AD!-PEG of the present disclosure, such as chimeric ADI-PEG 20, for treatment of a disease or condition of interest in accordance with teachings herein.
A pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, anoral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is generally either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystailine cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as aiginic acid, sodium alginate, Primogei, corn starch and the like; lubricants such as magnesium stearate or Sterotex; giidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer, in a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, in certain embodiments, physiological saline, Ringers solution, isotonic sodium chloride, fixed oils such as synthetic mono or digiycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethy!enediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of chimeric ADI as herein disclosed, such as chimeric ADI-PEG 20, such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 % of chimeric ADI in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of chimeric ADI-PEG. in certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of chimeric ADI-PEG prior to dilution.
The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emuisifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration, if intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition in solid or liquid form may include an agent that binds to chimeric AD!-PEG and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome. The pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers. and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.
The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a composition that comprises chimeric ADI-PEG as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-cova!entiy interact with the chimeric ADI-PEG composition so as to facilitate dissolution or homogeneous suspension of the chimeric ADI-PEG in the aqueous delivery system. The com positions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound (e.g., chimeric ADI-PEG) employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
A therapeutically effective amount of one of the compounds of the present invention is an amount that is effective to inhibit tumor growth. Generally, treatment is initiated with small dosages which can be increased by small increments until the optimum effect under the circumstances is achieved. Generally, a therapeutic dosage of compounds of the present invention may be from about 1 to about 200 mg/kg twice a week to about once every two weeks. For example, the dosage may be about 1 mg/kg once a week as a 2 ml intravenous injection to about 20 mg/kg once every 3 days. In a further embodiment, the dose may be from about 50 !U/m2 to about 8,000 IU/m2, administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks, in certain embodiments, the dose may be about 50 IU/m2, 60 !U/m2, 70 IU/m2, 80 !U/m2, 90 iU/m2, 100 IU/m2, 1 10 iU/rn2, 120 IU/m2, 130 IU/m2, 140 IU/m2, 150 IU/m2, 160 IU/m2, 170 IU/m2, 180 IU/m2, 190 IU/m2, 200 IU/m2, 210 IU/m2, 220 IU/m2, 230 IU/rn2, 240 IU/m2, 250 IU/rn2, 260 IU/m2, 270 !U/m2, 280 IU/m2, 290 IU/m2, 300 IU/m2, 310 IU/m2, about 320 IU/m2, about 330 IU/m2, 340 IU/m2 about 350 IU/m2, 360 IU/m2, 370 IU/m2, 380 !U/m2, 390 IU/m2, 400 !U/m2, 410 !U/m2, 420 IU/m2, 430 IU/rn2, 440 IU/m2, 450 IU/m2, 500 IU/m2, 550 IU/rn2, 600 IU/m2, 620 III/ rn2, 630 IU/m2, 640 IU/rn2, 650 !U/m2, 660 IU/m2, 670 !U/m2, 680 IU/m2, 690 !U/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, 850 IU/m2, 900 IU/m2, 950 IU/m2, 1 ,000 IU/m2, 1 ,100
U/m2, 1 ,200 IU/m2, 1 ,300 IU/m2 , 1 ,400 IU/m2, 1 ,500 IU/m2, 1 ,600 IU/m2 1 ,700 IU/m2 1 ,800
U/m2, 1 ,900 IU/m2, 2,000 IU/m2 , 2,100 IU/m2, 2,200 IU/m2, 2,300 IU/m2 2,400 IU/m2 2,500
U/m2, 2,600 IU/m2, 2,700 IU/m2, 2,800 IU/m2, 2,900 IU/m2, 3,000 IU/m2 3,100 IU/m2 3200
U/m2, 3,300 IU/m2, 3,400 IU/m2 , 3,500 IU/m2, 3,600 IU/m2, 3,700 IU/m2 3,800 IU/m2 3,900
U/m2, 4000 I U/m2, 4,100 I U/m2, 4,200 I U/m2, 4,300 I U/m2, 4,400 I U/m2, 4,500 U/m2, 4,600
U/m2, 4,700 IU/m2, 4,800 IU/m2 , 4,900 IU/m2, 5,000 IU/m2, 5,100 IU/m2 5,200 IU/m2 5,300
U/m2, 5,400 IU/m2, 5,500 IU/m2 , 5,600 IU/m2, 5,700 IU/m2, 5,800 IU/m2, 5,900 IU/m2 6,000
U/m2, 6, 100 IU/m2, 6,200 IU/m2 , 6,300 IU/m2, 6,400 IU/m2, 6,500 IU/m2 6,600 IU/m2 6,700
U/m2, 6,800 IU/m2, 6,900 IU/m2 , 7,000 IU/m2, 7,100 IU/m2, 7,200 IU/m2 7,300 IU/m2 7,400
U/m2, 7,500 IU/m2, 7,600 IU/m2 , 7,700 IU/m2, 7,800 IU/m2, 7,900 IU/m2 or about 8,000
I U/m1" administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks. In some embodiments, the dose may be about 1 rng/m2, 2 mg/m2. 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2. 60 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2 administered about once every 3 days, about once a week, about twice a week, or about once every 2 weeks, in certain embodiments, the dose may be modified as desired by the skilled clinician. In some embodiments,
The optimum dosage with chimeric ADi-SS-PEG5,000 may be about twice a week, while the optimum dosage with chimeric ADI-SS~PEG20,000 may be from about once a week to about once every two weeks. In certain embodiments, the optimum dosage with chimeric ADI-SS-PEG20,000 may be about twice a week.
Chimeric ADI-PEG may be mixed with a phosphate buffered saline solution, or any other appropriate solution known to those skilled in the art, prior to injection. In one embodiment, a liquid composition comprising chimeric ADI-PEG comprises about 10 to about 12 mg of chimeric ADI, about 20 to about 40 mg of polyethylene glycol, 1.27 mg +5% monobasic sodium phosphate, USP; about 3 mg +5% dibasic sodium phosphate, USP; 7.6 mg +5% sodium chloride, USP; at a pH of about 6.6 to about 7; in an appropriate amount of water for injection (e.g., about 1 ml or about 2 ml). In one embodiment, a liquid composition comprising a chimeric ADI-PEG comprises histidine - HCI, and in certain embodiments, the composition buffer is from about 0.0035M Histidine-HCi to about 0.35M Histidine-HCI. in one particular embodiment, the composition is formulated in a buffer comprising 0.035 M Histidine-HCI at pH 6.8 with 0.13 M sodium chloride, in another embodiment, the composition is formulated in a buffer comprising 0.02M sodium phosphate buffer at pH 6.8 with 0.13 M sodium chloride.
in one embodiment, a composition comprising chimeric ADI or chimeric ADI- PEG has a pH of about 5 to about 9, about 6 to about 8, or about 6.5 to about 7.5. in some embodiments, the composition comprising chimeric ADI has a pH of about 6.8 ± 1.0.
in one embodiment, free PEG in a composition comprising chimeric ADI-PEG is between 1-10%, and in a further embodiment, is less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1 % of the total PEG. in certain embodiments, the unmodified chimeric AD! in a composition comprising chimeric ADI-PEG is less than about 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less than 0.1 %. Generally, compositions comprising chimeric ADI-PEG have total impurities less than or equal to about 4%, 3%, 2%, 1.5%, 1 % or 0.5%. In one embodiment, the endotoxin limit meets the requirements stated in USP, i.e., < 50 EU/mL,
in one embodiment, the free sulfhydryi in a composition comprising chimeric ADI or chimeric ADI-PEG is greater than about 90%. In some embodiments, the free sulfhydryi in a composition comprising chimeric ADI or chimeric ADI-PEG is about 91 %, about 92%, about 93%, about 94% or about 95%, about 96% about 97%, about 98% about 99% or more.
in one embodiment, the chimeric AD! or chimeric ADI-PEG in a composition has a Km of from about 0.5 μΜ to about 15 μ , and in a further embodiment, is from about 1 μΜ to about 12 μΜ, about 1 μΜ to about 10 μΜ, about 1.5 μΜ to about 9 μ , about 1 .5 μΜ to about 8 μ or about 1.5 μΜ to about 7 μΜ. in certain embodiments, the chimeric ADi or chimeric ADI-PEG in a composition has a Km of about 1.5 μΜ to about 6.5 μΜ. In some embodiments, the chimeric ADi or chimeric ADI-PEG in a composition has a Km of about 1.5 μΜ, about 2 μΜ, about 2.5 μΜ, about 3 μΜ, about 3.5 μΜ, about 4 μΜ, about 4.5 μΜ, about 5 μ , about 5.5 μ , about 8 μΜ, about 6.5 μΜ, or about 7 μΜ. in one embodiment, the chimeric ADI or chimeric AD!-PEG in a composition has a reduced Km compared to a wild- type AD! or wiid-type ADI-PEG in the composition.
In one embodiment, the chimeric ADi or chimeric ADI-PEG in a composition has a Kcat of from about 0.5 sec"1 to about 15 sec"1, and in a further embodiment, is from about 1 sec"1 to about 12 sec"1, about 1 sec"1 to about 10 sec"1, about 1.5 sec"1 to about 9 sec"1, about 2 sec"1 to about 8 sec"1 or about 2.5 sec"1 to about 7 sec"1, in certain
embodiments, the chimeric ADI or chimeric ADI-PEG in a composition has a Kcat of about 2.5 sec"1 to about 7.5 sec"1, in some embodiments, the chimeric ADi or chimeric ADI-PEG in a composition has a Kcat of about 2.5 sec"1, about 3 sec"1, about 3.5 sec"1, about 4 sec"1, about 4.5 sec"1, about 5 sec"1, about 5.5 sec"1, about 6 sec"1, about 6.5 sec"1, about 7 sec"1, about 7.5 sec"1 or about 8 sec"1, in one embodiment, the chimeric ADI or chimeric ADI-PEG in a composition has a higher Kcat than a wild-type ADI or wild-type ADI-PEG in the composition.
in one embodiment, the chimeric AD! or chimeric ADI-PEG in a composition has a conductivity (also referred to in the art as specific conductance) of about 5 mS/cm to about 20 mS/cm, and in further embodiments, from about 5 mS/cm to about 15 mS/cm, about 7 mS/cm to about 15 mS/cm, about 9 mS/cm to about 15 mS/cm or about 10 mS/cm to about 15 mS/cm. In some embodiments, the chimeric ADI or chimeric ADI-PEG in a composition has a conductivity of about 9 mS/cm, about 10 mS/cm, about 1 1 mS/cm, about 12 mS/cm or about 13 mS/cm, about 14 mS/cm or about 15 mS/cm. In certain
embodiments, the chimeric ADi or chimeric ADI-PEG in a composition has a conductivity of about 13 mS/cm ± 1.0 mS/cm.
In one embodiment, the chimeric ADi or chimeric ADI-PEG in a composition has an osmolality of about 50 mOsm/kg to about 500 mOsm/kg, about 100 mOsm/kg to about 400 mOsm/kg, about 150 mOsm/kg to about 350 mOsm/kg, about 200 mOsm/kg to about 350 mOsm/kg or about 250 mOsm/kg to about 350 mOsm/kg. In certain embodiments, the chimeric AD! or chimeric ADI-PEG in a composition has an osmolality of about 300 ± 30 mOsm/kg,
in one embodiment, the protein concentration is about 1 1.0 ± 1 .0 mg/mL in certain embodiments, the protein concentration is between about 8 and about 15 mg/mL, In another embodiment, the protein concentration is about 8, 9, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 13, 13.5, 14, or 15 mg/mL.
in one embodiment, the specific enzyme activity is between 5.0 and 150 !U/mg, where 1 IU is defined as the amount of enzyme that converts one moi of arginine into one mol of citrulline and 1 μρηο! of ammonia in one minute at 37° C and the activity is 100 ± 20 iU/mg. In another embodiment, the specific enzyme activity is about 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.0, 9.5, 10, 10.5, 1 1 , 1 1 .5, 12, 12,5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, or about 150 ± 2.0 IU/mg. In one particular embodiment, the specific enzyme activity is 100 ± 10.0 IU/mg.
Compositions comprising chimeric ADI-PEG of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising chimeric ADI-PEG (e.g., chimeric ADI-PEG 20) of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, chimeric ADI-PEG as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, chimeric ADI-PEG as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising chimeric ADi-PEG and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
Thus, in certain embodiments, also contemplated is the administration of the chimeric ADI compositions of this disclosure in combination with one or more other therapeutic agents, Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer or GVHD. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-infiammatories, chemotherapeutics, radiotherapeutics, autophagy modulators, or other active and ancillary agents.
In certain embodiments, the chimeric AD! compositions disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); aikyi sulfonates such as busuifan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethyleneme!amine, trietylenephosphoramide, triethyienethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorefhamine, mechiorefharnine oxide hydrochloride, meipha!an, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, !omustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marceilomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fiuorouracii (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocifabine, floxuridine, 5~FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiosfane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as froiinic acid; aceg!atone; a!dophosphamide glycoside; aminolevulinic acid; amsacrine;
bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyliinic acid; 2-ethyihydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran;
spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichiorotriethyiamine; urethan;
vindesine; dacarbazine; mannomustine; mitobronitoi; mito!actol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paciitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ.) and docetaxei (TAXOTERE®., Rhne- Pouienc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carbop!atin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; dif!uoromethyiomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (aiitretinoin) ; ONTAK™ (deni!eukin diftitox) ; esperamicins; capecitabine; and pharmaceuticaiiy acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoies, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as f!utamide, ni!utamide, bica!utamide, ieuproiide, and goserelin. Further chemotherapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib. bevacizumab, cetuximab, crizotinib, dasatinib, eriotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxoiitinib, trastuzumab, vandetanib, vemurafenib, and sunitinib; siroiimus (rapamycin), everolimus and other mTOR inhibitors. Pharmaceutically acceptable salts, acids or derivatives of any of the above are also contemplated for use herein.
in certain embodiments, the chimeric ADI compositions disclosed herein may be administered in conjunction with any number of autophagy inhibitors, in some preferred embodiments, the autophagy inhibitor is selected from the group consisting of: chloroquine, 3-methyiadenine, hydroxychloroquine (Plaquenil.TM.), bafilomycin A1 , 5-amino-4-imidazoie carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1 , analogues of cAMP, and drugs which elevate cAMP levels, adenosine, N6-mercaptopurine riboside, wortmannin, and vinblastine, in addition, antisense or siRNA that modulates expression of proteins essential for autophagy, such as for example ATG5, may also be used.
in one embodiment, the combination of chimeric ADI-PEG with one or more therapeutic agents acts additively or synergisticaily. In this regard, synergizing agents are described herein, which include a therapeutic agent (e.g., chemotherapeutic agent, autophagy inhibitor, mTOR inhibitor, or any other therapeutic agent used for the treatment of cancer, GVHD, or inflammatory bowel disease as described herein) that is capable of acting synergisticaily with chimeric ADI-PEG as provided herein, where such synergy manifests as a detectable effect that is greater (i.e., in a statistically significant manner relative to an appropriate control condition) in magnitude than the effect that can be detected when the chemotherapeutic agent is present but the ADi-PEG composition is absent, and/or when the ADI-PEG is present but the chemotherapeutic agent is absent. Methods for measuring synergy are known in the art (see e.g., Cancer Res January 15, 2010 70; 440). The com positions comprising chimeric ADi , and optionally other therapeutic agents, as described herein may be used in therapeutic methods for treating of cancer and methods for preventing metastasis of a cancer. Thus, the present invention provides for methods for treating, ameliorating the symptoms of, or inhibiting the progression of or prevention of a variety of different cancers, in another embodiment, the present disclosure provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of GVHD. In particular the present disclosure provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer or GVHD in a patient comprising administering to the patient a therapeutically effective amount of chimeric ADi composition as described herein, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer or GVH D. Thus, the chimeric ADI compositions described herein may be administered to an individual afflicted with inflammatory bowel disease (e.g., Crohn's disease; ulcerative colitis), GVHD or a cancer, including, but not limited to leukemia (e.g. acute myeloid leukemia and relapsed acute myeloid leukemia), melanoma, sarcomas (including, but not limited to, metastatic sarcomas, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as, but not limited to, hormone refractory prostate cancer), mesothelioma, lymphatic leukemia, chronic myelogenous leukemia, lymphoma, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including, but not limited to, gastric adenocarcinoma), glioma, glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney cancer (including but not limited to renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers (including, but not limited to, squamous cell carcinoma of the head and neck; cancer of the tongue), cervical cancer, testicular cancer, gallbladder,
cholangiocarcinoma, and stomach cancer.
In one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of myeloid leukemia, such as, but not limited to, acute myeloid leukemia (A L), by administering a therapeutically effective amount of a chimeric ADI-PEG 20. in certain embodiments, the myeloid leukemia, such as AML, is deficient in ASS, ASL, or both. In a further embodiment, the present disclosure provides a method of treating AML comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks, in certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of AML is between about 50 ! U/m2 and about 8,000 I U/m'-, and in other embodiments is about 50 I U/m2, about 100 I U/rn2, 150 I U/m2, 200 I U/m2, 250 I U/m2, 300 I U/m2, 350 l U/m2, 400 I U/m2, 450 I U/m2, 500 I U/m2, 550 I U/m2, 600 I U/m2, 650 I U/m2, 700 I U/m2, 750 I U/m2, 800 I U/m2, about 900 i U/m2, about 1 ,000 I U/m2, 1 ,500 I U/m2 about 2,000 I U/m2, about 2,500 IU/m2, about 3,000 iU/rn2, 3,500 IU/m2, 4,000 !U/m2, 4,500 IU/m2, 5,000 iU/rn2, 5,500 IU/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 IU/m2. In particular embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of AML is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 60 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating AML, wherein the dose of chimeric ADI is doubled and may be increased to 640 !U/m2 per week or more. In one particular embodiment the chimeric AD! for the treatment of AML is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI. in another embodiment, the present disclosure provides a method of treating AML by administering a composition comprising chimeric ADI-PEG 20 wherein the composition comprises a chimeric ADI modified with 5±1.5 PEG molecules, and in one embodiment, 5±1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., unmodified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of sarcomas, including but not limited to metastatic sarcomas, by administering a therapeutically effective amount of a chimeric ADI-PEG 20. In certain embodiments, the sarcoma is deficient in ASS, ASL, or both. In a further embodiment, the present disclosure provides a method of treating a sarcoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks. In certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of sarcomas is between about 50 IU/m2 and about 8,000 IU/m2, and in other embodiments is about 50 IU/m2, about 100 IU/m2, 150 !U/m2, 200 IU/m2, 250 IU/m2, 300 IU/m2, 350 IU/m2, 400 IU/m2, 450 IU/m2, 500 IU/m2, 550 IU/m2, 600 IU/m2, 650 IU/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, about 900 IU/m2, about 1 ,000 IU/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 IU/m2, about 3,000 IU/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 IU/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/rn2, or about 8,000 IU/m2. in particular embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of sarcomas is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 60 mg/m2. 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating sarcoma, wherein the dose of chimeric ADi is doubled and may be increased to 640 IU/m2 per week or more. In one particular embodiment the chimeric ADi for the treatment of AML is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI. In another embodiment, the present disclosure provides a method of treating a sarcoma, including a metastatic sarcoma, by administering a composition comprising chimeric ADI-PEG 20 wherein the composition comprises an chimeric ADi modified with 5±1.5 PEG molecules, and in one embodiment, 5±1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADi (i.e., unmodified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of pancreatic cancer by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with an autophagy inhibitor, such as but not limited to chloroquine, 3- methyiadenine, hydroxychloroquine, bafilomycin A1 , 5-amino-4-imidazoie carboxamide riboside (AICAR), okadaic acid, N6-mercaptopurine riboside, wortmannin, and vinblastine, in certain embodiments, the pancreatic cancer is deficient in ASS, ASL or both, in a further embodiment, the present disclosure provides a method of treating pancreatic cancer comprising administering ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of an autophagy inhibitor, such as chloroquine. in this regard, a
therapeutically effective dose of chloroquine may be an initial dose of about 800 mg base followed by an additional 300 mg base and a single dose of 300 mg base on each of two consecutive days. This represents a total dose of 2.5 g chloroquine phosphate or 1.5 g base in three days. In further embodiments, the dose may be about 300 mg base. The dose of chloroquine, or other autophagy inhibitor, may be modified as needed by a skilled clinician using dosages known in the art. As would be understood by the skilled person, the autophagy inhibitor may be administered before, at the same time as or after a composition comprising ADI-PEG 20. In certain embodiments, the dose of ADI-PEG 20 administered for the treatment of pancreatic cancer is between about 50 iU/m2 and about 8,000 IU/m2, and in other embodiments is about 50 IU/m2, about 100 IU/m2, 150 IU/m2, 200 IU/m2, 250 IU/m2, 300 IU/m2, 350 IU/m2, 400 IU/m2, 450 IU/m2, 500 IU/m2, 550 IU/m2, 600 IU/m2, 650 IU/m2, 700 IU/m2, 750 Mm2, 800 IU/m2, about 900 IU/m2, about 1 ,000 IU/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 IU/m2, about 3,000 IU/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 IU/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 I U/m2. In particular embodiments, the dose of ADi-PEG 20 administered for the treatment of pancreatic cancer is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m^, 8 mg/m2, 9 mg/m , 10 mg/m2, 15 mg/m , 20 mg/m2, 25 rng/rn^, 30 mg/m2, 35 mg/m*, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 80 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating pancreatic cancer, wherein the dose of chimeric ADI is doubled and may be increased to 640 I U/m2 per week or more. In one particular embodiment the chimeric ADI for the treatment of pancreatic cancer is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI. In another embodiment, the present disclosure provides a method of treating pancreatic cancer by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with chloroquine, or other appropriate autophagy inhibitor, wherein the composition comprises a chimeric ADI modified with 5±1 .5 PEG molecules, and in one embodiment, 5±1 .5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of small cell lung cancer by administering a therapeutically effective amount of ADI-PEG 20, optionally in combination with an autophagy inhibitor. In certain embodiments, the small ceil lung cancer is deficient in ASS, ASL, or both. In a further embodiment, the present disclosure provides a method of treating small ceil lung cancer comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks;
optionally in combination with a therapeutically effective amount of an autophagy inhibitor, such as chloroquine. in this regard, a therapeutically effective dose of chloroquine may be an initial dose of about 800 mg base followed by an additional 300 mg base and a single dose of 300 mg base on each of two consecutive days. This represents a total dose of 2.5 g chloroquine phosphate or 1 .5 g base in three days, in further embodiments, the dose may be about 300 mg base. The dose of chloroquine may be modified as needed by a skilled clinician using dosages known in the art. As would be understood by the skilled person, the autophagy inhibitor may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20. In certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of small cell lung cancer is between about 50 I U/m2 and about 8,000 I U/m2, and in other embodiments is about 50 ! U/m2, about 100 I U/m2, 150
I U/m2, 200 I U/m2, 250 I U/m2, 300 I U/m2, 350 I U/m2, 400 I U/m2, 450 I U/m2, 500 I U/m2, 550 IU/m2, 600 IU/m2, 650 IU/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, about 900 !U/m2, about 1 ,000 IU/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 IU/m2, about 3,000 IU/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 !U/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 IU/m2. In particular embodiments, the dose of chimeric ADI- PEG 20 administered for the treatment of small cell lung cancer is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 60 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating small cell lung cancer, wherein the dose of chimeric ADi is doubled and may be increased to 640 IU/m2 per week or more. In one particular embodiment the chimeric AD! for the treatment of small cell lung cancer is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI. In another embodiment, the present disclosure provides a method of treating small ceil lung cancer by administering a composition comprising chimeric ADi - PEG 20 optionally in combination with chloroquine, wherein the composition comprises an chimeric ADI modified with 5±1.5 PEG molecules, and in one embodiment, 5±1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of sarcomas (including but not limited to, metastatic sarcomas) by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with an autophagy inhibitor. In certain embodiments, the sarcoma is deficient in ASS, ASL, or both, in a further embodiment, the present disclosure provides a method of treating sarcoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of an autophagy inhibitor, such as chloroquine. In this regard, a therapeutically effective dose of chloroquine may be an initial dose of about 600 mg base followed by an additional 300 mg base and a single dose of 300 mg base on each of two consecutive days. This represents a total dose of 2.5 g chloroquine phosphate or 1.5 g base in three days, in further
embodiments, the dose may be about 300 mg base. The dose of chloroquine may be modified as needed by a skilled clinician using dosages known in the art. As would be understood by the skilled person, the autophagy inhibitor may be administered before, at the same time as or after a composition comprising ADI-PEG 20, In certain embodiments, the dose of ADI-PEG 20 administered for the treatment of sarcoma is between about 50 IU/m2 and about 8,000 IU/m2, and in other embodiments is about 50 !U/m2, about 100 IU/m2, 150 IU/m2, 200 IU/m2, 250 IU/m2, 300 IU/m2, 350 IU/m2, 400 IU/m2, 450 IU/m2, 500 !U/m2, 550 !U/m2, 800 IU/m2, 650 IU/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, about 900 iU/m2, about 1 ,000 IU/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 IU/m2, about 3,000 !U/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 IU/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 IU/m2. in certain embodiments, the dose of ADI-PEG 20 administered for the treatment of sarcoma is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 60 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating sarcoma, wherein the dose of chimeric ADI is doubled and may be increased to 640 IU/m2 per week or more, in one particular embodiment the chimeric ADi for the treatment of sarcoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI. In another embodiment, the present disclosure provides a method of treating sarcoma by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with chloroquine, wherein the composition comprises a chimeric ADI modified with 5±1 .5 PEG molecules, and in one embodiment, 5±1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of melanoma by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with docetaxei. In certain embodiments, the melanoma is deficient in ASS, ASL, or both, in a further embodiment, the present disclosure provides a method of treating melanoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of docetaxei. In this regard, a therapeutically effective dose of docetaxei may comprise 75mg/m2 or 100 mg/m2 administered intravenously over between 30 minutes and 1 hour about every 3 weeks. As would be understood by the skilled clinician, the dose of docetaxei may be modified depending on disease indication and/or prior treatments, and docetaxei may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20. in certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 50 IU/m and about 8,000 IU/m2, and in other embodiments is about 50 !U/m2, about 100 iU/m2, 150 iU/m2, 200 IU/m2, 250 IU/m2, 300 iU/m2, 350 IU/m2, 400 IU/m2, 450 IU/m2, 500 IU/m2, 550 IU/m2, 600 IU/m2, 650 IU/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, about 900 IU/m2, about 1 ,000 IU/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 IU/m2, about 3,000 IU/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 IU/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 IU/m2. the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 60 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. in certain embodiments, the present disclosure provides a method of treating melanoma, wherein the dose of chimeric ADI is doubled and may be increased to 640 IU/m2 per week or more. In one particular embodiment the chimeric ADI for the treatment of melanoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5, PEG molecules per chimeric ADI. In another embodiment, the present disclosure provides a method of treating melanoma by a composition comprising chimeric ADI-PEG 20, optionally in combination with docetaxei, wherein the composition comprises an chimeric ADI modified with 5±1.5 PEG molecules, and in one embodiment, 5±1.5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. In a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of melanoma by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with cisplatin. In certain embodiments, the melanoma is deficient in ASS, ASL, or both. In a further embodiment, the present disclosure provides a method of treating melanoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks; optionally in combination with a therapeutically effective amount of cisplatin. in this regard, a therapeutically effective dose of cisplatin may comprise administration either once per cycle (every 3-4 weeks) at 50-100 mg/m2, or daily for 5 days for a total of 100 mg/m2 per cycle. As would be understood by the skilled clinician, the dose of cisplatin may be modified depending on disease indication, individual patient, and/or prior treatments, and cisplatin may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20. In certain
embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 50 IU/m2 and about 8,000 IU/m2, and in other embodiments is about 50 IU/m2, about 100 IU/m2, 150 IU/m2, 200 IU/m2, 250 IU/m2, 300 IU/m2, 350 IU/m2, 400 IU/m2, 450 IU/m2, 500 !U/m2, 550 IU/m2, 600 IU/m2, 650 IU/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, about 900 IU/m2, about 1 ,000 IU/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 IU/m2, about 3,000 IU/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 IU/m2, 8,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 IU/m2, In certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 1 mg/m2 and about 80 mg/m2 and in other embodiments is about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2. 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2, 55 mg/m2, 60 mg/m2. 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating melanoma, wherein the dose of chimeric ADI is doubled and may be increased to 640 IU/m2 per week or more. In one particular embodiment the chimeric ADI for the treatment of melanoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5, PEG molecules per chimeric ADI. in another embodiment, the present disclosure provides a method of treating melanoma by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with cisplatin, wherein the composition comprises a chimeric ADI modified with 5±1.5 PEG molecules, and in one embodiment, 5±1 ,5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADI (i.e., not modified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
in one embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of renal ceil carcinoma by administering a therapeutically effective amount of chimeric ADI-PEG 20, optionally in combination with an mTOR inhibitor, such as but not limited to rapamycin, temsirolimus, everolimus, and ridaforo!imus. in certain embodiments, the renal cell carcinoma is deficient in ASS, ASL, or both. In a further embodiment, the present disclosure provides a method of treating renal cell carcinoma comprising administering chimeric ADI-PEG 20 about once every 3 days, about once a week, about twice a week, or about once every 2 weeks;
optionally in combination with a therapeutically effective amount of an mTOR inhibitor, such as rapamycin. The dose of rapamycin, or other mTOR inhibitor, may be determined as needed by a skilled clinician using dosages known in the art. As would be understood by the skilled person, the mTOR inhibitor may be administered before, at the same time as or after a composition comprising chimeric ADI-PEG 20. in certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of renal cell carcinoma is between about 50 IU/m2 and about 8,000 IU/m2, and in other embodiments is about 50 IU/m2, about 100 IU/m2, 150 !U/m2, 200 IU/m2, 250 IU/m2, 300 IU/m2, 350 IU/m2, 400 IU/m2, 450 IU/m2, 500 IU/m2, 550 IU/m2, 600 IU/m2, 650 IU/m2, 700 IU/m2, 750 IU/m2, 800 IU/m2, about 900 IU/m2, about 1 ,000 !U/m2, 1 ,500 IU/m2 about 2,000 IU/m2, about 2,500 !U/m2, about 3,000 IU/m2, 3,500 IU/m2, 4,000 IU/m2, 4,500 IU/m2, 5,000 IU/m2, 5,500 IU/m2, 6,000 IU/m2, 6,500 IU/m2, 7,000 IU/m2, 7,500 IU/m2, or about 8,000 IU/m2. In certain embodiments, the dose of chimeric ADI-PEG 20 administered for the treatment of melanoma is between about 1 mg/m2, 2 mg/m2, 3 mg/m2, 4 mg/m2, 5 mg/m2, 6 mg/m2, 7 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 15 mg/m2, 20 mg/m2, 25 mg/m2, 30 mg/m2, 35 mg/m2, 40 mg/m2, 45 mg/m2, 50 mg/m2. 55 mg/m2, 60 mg/m2, 65 mg/m2, 70 mg/m2, 75 mg/m2, or about 80 mg/m2. In certain embodiments, the present disclosure provides a method of treating renal cell carcinoma, wherein the dose of chimeric ADi is doubled and may be increased to 640 IU/m2 per week or more. In one particular embodiment the chimeric ADI for the treatment of renal cell carcinoma is modified with 3.5-6.5, or in one embodiment, 4.5 - 5.5 PEG molecules per chimeric ADI. In another embodiment, the present disclosure provides a method of treating renal cell carcinoma by administering a composition comprising chimeric ADI-PEG 20, optionally in combination with rapamycin, or other appropriate mTOR inhibitor, wherein the composition comprises a chimeric ADi modified with 5±1.5 PEG molecules, and in one embodiment, 5±1 ,5 straight chain PEG molecules, and, in certain embodiments, the composition comprises less than about 0.5% native chimeric ADi (i.e., not modified with PEG) and/or less than about 5% free PEG. in a further embodiment, the composition comprises a histidine - HCL buffer.
The present disclosure also provides methods of treating, ameliorating the symptoms of, or inhibiting the progression of an inflammatory disorder in a patient comprising administering to the patient a composition comprising chimeric ADI (e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20), as described herein, alone or in combination with one or more other therapeutic agents. In one embodiment, the present disclosure also provides methods of treating, ameliorating the symptoms of, or inhibiting the progression of an inflammatory bowel disease in a patient comprising administering to the patient a composition comprising chimeric ADI (e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20), as described herein, alone or in combination with one or more other therapeutic agents. In this regard, the present disclosure provides methods of treating, ameliorating the symptoms of, or inhibiting the progression of Crohn's disease or ulcerative colitis in a patient comprising administering to the patient a composition comprising chimeric ADI (e.g., chimeric ADI-PEG, in particular chimeric ADI-PEG 20), as described herein, alone or in combination with one or more other therapeutic agents.
58 in another embodiment, the present disclosure provides a method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer in a patient comprising administering to the patient a composition comprising chimeric ADI, and optionally one or more other therapeutic agents, as described herein, wherein the cancer is deficient in ASS, ASL, or both. In this regard, ASS or ASL deficiency may be a reduction in expression as measured by mRNA expression or protein expression, or may be a reduction in protein activity, and generally comprises a statistically significant reduction in expression or activity as determined by the skilled person. Reduced ASS or ASL expression or activity may be a reduction in expression or activity of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more, as compared to expression or activity in an appropriate control sample known to be cancer free. In certain embodiments, ASS or ASL expression or activity is reduced by at least twofold as compared to expression or activity in a non-cancer control sample.
in certain embodiments, the reduced expression or activity of ASS or ASL results from methyiation of the ASS or ASL promoter. In another embodiment the reduction in expression or activity of ASS or ASL results from a DNA mutation (e.g., one or more point mutations, small deletions, insertions, and the like) or a chromosomal abnormality resulting in deletion of the gene, in one embodiment, the cancer is ASS or ASL negative, meaning no expression or activity is observed.
Reduction in ASS or ASL expression or activity may be measured using any methods known in the art, such as but not limited to, quantitative PCR,
immunohistochemistry, enzyme activity assays (e.g., assay to measure conversion of cifruliine into argininosuccinafe or conversion of argininosuccinate into arginine and fumarate), and the like.
Thus, the present invention provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of a cancer in a patient comprising administering to the patient a composition comprising chimeric ADI as described herein, wherein the cancer exhibits reduced expression or activity of ASS or ASL, or both, wherein the cancer includes, but is not limited to leukemia (e.g. acute myeloid leukemia and relapsed acute myeloid leukemia), melanoma, sarcomas (including, but not limited to, metastatic sarcomas, uterine leiomyosarcoma), pancreatic cancer, prostate cancer (such as, but not limited to, hormone refractory prostate cancer), mesothelioma, lymphatic leukemia, chronic
myelogenous leukemia, lymphoma, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer (including, but not limited to, gastric adenocarcinoma), glioma, glioblastoma multi-form, retinoblastoma, neuroblastoma, non-small cell lung cancer (NSCLC), kidney cancer (including but not limited to renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers (including, but not limited to, squamous ceil carcinoma of the head and neck; cancer of the tongue), cervical cancer, testicular cancer, gallbladder, cholangiocarcinorna, and stomach cancer.
Various studies in the literature have shown that ASS is deficient in the following tumors:
Figure imgf000059_0001
Accordingly, treatment of these ASS-deficient cancers is specifically contemplated herein, with chimeric ADI-PEG alone or in combination with other treatments.
The present invention further provides methods for treating, ameliorating the symptoms of, or inhibiting the progression of cancer in a patient comprising administering to the patient a composition comprising chimeric ADI as described herein (e.g. , chimeric ADI- PEG and in particular chimeric ADI-PEG 20), in combination with an autophagy inhibitor. In one embodiment, the present invention provides methods for treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a composition comprising chimeric ADI as described herein in combination with autophagy inhibitor wherein the cancer is pancreatic cancer or small ceil lung cancer. in certain embodiments, the present invention provides methods of treatment where administration of the compositions comprising chimeric AD! described herein depletes arginine in the plasma for at least one month, 2 months, 3 months, 4 months, 5 months, 6 months or longer.
EXAMPLES
EXAMPLE 1
CHIMERIC ADI ENZYMES ARE ACTIVE AND LESS CROSS-REACTIVE WITH PATIENT ANTI-ADI-PEG
20 ANTIBODIES
This Example describes the generation of artificially engineered chimeric ADI enzymes composed of (1 ) protein with arginine deiminase enzymatic activity, (2) reduced cross reactivity with anti-ADI-PEG 20 antibodies, (3) reduced number of lysine residues, and/or (4) PEG conjugation with chemically stable linkers.
ADI Preparation. Recombinant chimeric ADI enzymes were cloned, expressed, and purified for testing according to standard protocols, as described, for example, in Gailego et a!., PLOS One, 7(10):e47886, 2012; Monstadt and Holidorf, Biochem. J. 273:739-745, 1990; Joo Noh et a!., Molecules and Cells. 13:137-143, 2002; and Sugimura et a!., Infection and Immunity. 58:2510-2515, 1990. See Table A1 for the amino acid sequences of the chimeric ADI enzymes.
Human Anti-ADI-PEG2Q Antibody Purification. Anti-ADi-PEG20 antibody was purified from plasma samples of patients who had received ADI-PEG20 during a clinical study. A total of 60 ml of plasma was pooled from 8 different patients that had reached high titer (titer >/= 4) against ADI-PEG20 as determined by an ELISA assay. A two-step purification was used, a Protein "A" chromatography (GE Healthcare) followed by an ADI affinity chromatography. -20 mg of purified antibody was obtained and stored at -80°C in aiiquots until needed.
API Enzyme Assays. Arginine deiminase (ADI) catalyzes the conversion of L- arginine to L-citruiline and ammonia. The amount of L-citrul!ine can be detected by a colorimetric endpoint assay (see, for example, Knipp and Vasak, Analytical Biochem.
288:257-264, 2000) and compared to a standard curve of known amounts of L-citruiline in order to calculate the specific activity of ADI expressed as iU/mg of protein. One IU of enzyme activity is defined as the amount of enzyme that produces 1 μηιοΙ of citru!line per minute at the pH and temperature being tested. Standard assay conditions were performed at 37°C in Physiological HEPES Buffer (PHB) 50 mM HEPES, 180 mM NaCI pH 7.4 (Lang and Zander, Clin Chem Lab Med. 37:563-571 , 1999) plus 0.1 % BSA. All samples and standards were run in duplicate or triplicate where conditions permitted.
Km and Kcat values were determined by using a variation of the activity assay described above. As with the activity assay, all reactions were run at 37°C in PHB plus 0.1 % BSA. Enzyme concentration, reaction time, and substrate concentration range were adjusted for each of the ADI or ADIr constructs to account for their differences in activity, in general, 2 nM enzyme, 5 minute reaction time, and a 0 - 160 μΜ arginine was used as starting conditions. When optimizing the conditions, particular attention was paid towards the amount of substrate consumed as a percentage of total substrate added to the reaction. The lower limit of detection is 1 μΜ of citruiline with the lower limit of quantitation being 2 μΜ. A citruliine standard curve was run on every plate and used to quantify the citruliine produced by the enzymatic reaction.
Calculations. The citruiline concentration (μΜ) produced in each reaction well was calculated and averaged using the citruliine standard curve. The velocity of each reaction was then calculated in M/min/SO nM ADI. Specific activity (!U/mg or mois product min/mg ADI) was calculated by multiplying this value by the "III" factor (IU factor was calculated from the molecular weight of the ADI and the reaction volume).
Arginine deiminase enzymatic activity. The results of the ADI enzyme assays are shown in Table E1 .
Figure imgf000061_0001
table LI . C ssf sersc A OS nayines.
SLQ ID Sa&cffic Nama Catalytic sJotrsam c-riesfcai ϋοίΐ)» »
HO: Activity
M. columhinum M. arthritidis
C4DS3 30
(1-67, 148-401 ) (75-152)
M. co!umbinum M. gateae
C4DS5 31 +++
{1-67, 148-401 ) (75-152)
A columbinum M. phocicerebrale
C4DS6 32 ++
{1-67, 148-401 ) (75-152)
M. columbinum M. phocidae
C4DS7 33 n.d.
{1-67, 148-401 ) (75-152)
M. columbinum . galiinarum
C4DS8 34
(1-67, 148-401 ) (68-147)
M, columbinum M. ners
C4DS9 35 +++++
{1-67, 148-401 ) (68-147)
Af. gateae M. ailigatoris
C5DS1 36 ++÷
(1-74, 153-410) (71-148)
M. gateae M. arginini
C5DS2 37 ++÷+
(1-74, 153-410) (75-152)
M. gateae M. arthritidis
C5DS3 38 +++
(1-74, 153-410) (75-152)
M, oateae , columbinum
C5DS4 39 +++
(1-74, 153-410) (68-147)
A/f. gateae M. phocicerebrale
C5DS6 40 +++
(1-74, 153-410) (75-152)
M. gateae phocidae
C5DS7 41 ++■·■
(1-74, 153-410) (75-152)
M. phocicerebrale M. aliiqatoris
C6DS1 42 n.d.
( 1-74, 153-410) (71-148)
M, phocicerebrale , arginini
C6DS2 43 ++■+■+
(1-74, 153-410) (75-152)
A/f. phocicerebrale M. arthritidis
C6DS3 44 +
(1-74, 153-410) (75-152)
A phocicerebrale Af. columbinum
C6DS4 45 n.d.
(1-74, 153-410) (68-147)
M. phocicerebrale M. gafeae
C6DS5 46 ++÷
( 1-74, 153-410) (75-152)
M. phocicerebrale M. phocidae
C6DS7 47 +++
(1-74, 153-410) (75-152)
M. phocidae M. alligatoris
C7DS1 48 n.d.
(1-74, 153-410) (71-148)
A phocidae A arginini
C7DS2 49 ++■·■■·■
(1-74, 153-410) (75-152)
M. phocidae M. arthritidis
C7DS3 50 ++÷
(1-74, 153-410) (75-152)
M. phocidae M. columbinum
C7DS4 51 +++
(1-74, 153-410) (68-147)
M. phocidae M. gateae
C7DS5 52 +++
{1-74, 153-410) (75-152)
A phocidae A phocicerebrale
C7DS6 53
(1-74, 153-410) (75-152)
M. galiinarum A arthritidis
C8DS3 54 n.d.
(1-67, 148-401 ) (75-152)
M. galiinarum M. columbinum
C8DS4 55 +++++
(1-67, 148-401 ) (68-147)
M, galiinarum , /nere
C8DS9 56 +++++
(1-67, 148-401 ) (68-147) table El . C ssf sersc A
SEQ ID Sp&cific Nama Catalytic Dotrsam e-HeHcai Doinasn
HO: Activity
M. iners M. arthritidis
C9DS3 57 n.d.
(1-67, 148-401 ) (75-152)
M. iners M. co!umbinum
C9DS4 58 +++++
(1-87, 148-401 ) (68-147)
M. iners M. galiinarurn
C9DS8 59
(1-67, 148-401 ) (68-147)
Chimeric moiecuies were engineered from M. arginini (SEQ ID NO:2), M. arthritidis (SEQ ID
NO:3), and hominis (Phoenix sequence set forth in SEQ ID NO: 14, with modifications). The parenthetical numbers specify the amino acid residues from the natural enzymes, used to form the domains. A C-terminal tryptophan was added to the published M. arthritidis sequence and mutation C251 S was made for M. arginini and M. arthritidis. The ADI specific activity (lU/mg) of these non-pegylated enzymes is shown relative to ADi-PEG 20 (+++).
The results in Table E1 show that the engineered chimeric ADI enzymes described herein have efficient catalytic activity. The catalytic parameters Km and kcat for these enzymes is sufficient to remove arginine and maintain low arginine concentrations in the blood. These parameters are preferably less than 20 μΜ and greater than 1 sec" 1 , respectively. The pH optimum is around 7.4 so as to maintain efficient catalytic activity in blood. The enzyme stability, as well as that of the covalentiy linked PEG, is such that it should be maintained during long-term storage and patient treatment at 37°C.
Reduced cross reactivity with anti-ADI-PEG 20 antibodies. ADI is composed of two domains, a catalytic domain and an ohelical domain. The present invention is directed in part to engineered, artificial, chimeric, recombinant enzymes with ADI activity. Each is composed of two domains, where each domain is selected from a number of possible species. Domain boundaries are determined by examination of ADI X-ray crystal structures from M. hominis and M. arginini, and extending this to other Mycoplasma ADI enzymes by homology.
The use of domains from different species of ADI enzymes can maintain the catalytic activity while changing a number of surface residues. Some of these surface residues form epitopes for anti-ADI-PEG 20 antibodies developed during a patient's treatment with ADi-PEG 20. Their replacement can reduce the antigenicity with respect to anti-ADI-PEG 20 antibodies, therefore reducing anti-ADI-PEG 20 antibody neutralization and clearance of the modified drug. This is shown in Table E2, where two preparations of anti- ADI-PEG 20 antibodies showed less binding to the DS1 , DS2, DS3, and DS4 antigens, compared to M. hominis ADI . This can be attributed to residue changes on the surface of these antigens that alter the epitopes and disrupt antibody-antigen binding interactions. Table E2. Reduction of Amts-ADS-PEG 20 Antibody Binding to ADS Arstigerss Compared to
AD1-PEG 20
Antibody Antigens
Cone DS1 DS2 DS3 DS4 pg/m 1 g/mL 1 g/mL 1 pg/mL 1 pg/mL L
Human anti-ADI-PEG 20 IgG (titer 3) 10,0 Yes No Yes No
Human anti-ADI-PEG 20 IgG (titer 3) 25.0 Yes No Yes No
Human anti-ADI-PEG 20 IgG (titer 3) 50.0 Yes Yes Yes Yes
Human anti-ADI-PEG 20 IgG (titer 4) 0.5 Yes No Yes No
Human anti-ADI-PEG 20 IgG (titer 4) 1.0 Yes No Yes No
Human anti-ADI-PEG 20 IgG (titer 4) 5.0 Yes Yes Yes Yes
Surface Lysine Residue Content Reduction. The arginini (catalytic domain) - M. arthritidis (a-heiica! domain) chimeras were further modified by replacing surface lysine residues with amino acid residues other than lysine and monitoring ADi activity. Four mutants were made (Table E3) and their ADI activity was determined.
Figure imgf000064_0001
Table E4 shows ADI activity of the DS1 (M. arginini - M. arthritidis) enzyme and 4 lysine replacement mutants. Lysine reduction was undertaken to reduce the number of potential pegy!ation sites.
Figure imgf000064_0002
With around 30 potential pegylation sites, the PEG occupancy is generally small at each site. Reducing the number of potential pegylation sites will result in higher PEG occupancies and more complete shielding at each remaining site. This is expected to increase proteolytic protection and reduce immune cross reactivity to affinity matured anti- ADi-PEG 20 antibodies from previous treatments. It will also produce a more uniform drug.
In summary, the present Examples describes engineered ADl enzymes with excellent ADl activity, with anti-ADi-PEG 20 antibody epitopes removed to reduce antibody neutralization and clearance, and with protection from proteolysis and renal clearance by pegylation.
The various embodiments described above can be combined to provide further embodiments. Ail of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description, in general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A recombinant chimeric arginine deiminase (ADI) comprising a catalytic domain oi an AD! protein derived from a first microorganism and an a-heiical domain of an ADI protein derived from a second microorganism.
2. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is selected from the genera Mycoplasma, Clostridium, Bacillus, Borrelia, Enterococcus, Streptococcus, Lactobacillus, and Giardia.
3. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is selected from the group consisting of Mycoplasma pneumonia, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Streptococcus pyogenes, streptococcus pneumonia, Borrelia burgdorferi, Borrelia afzeiii, Giardia intestinaiis, Clostridium perfringens, Bacillus licheniformis, and Enterococcus faecalis.
4. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is selected from the group consisting of M. arginini, M. arthritidis, M, hominis,
Mycoplasma pneumonia, Mycoplasma phocicerebrale, Mycoplasma oraie,
Mycoplasma gateae, Mycoplasma phocidae, Mycoplasma columbinum, Mycoplasma iowae, Mycoplasma crocodyli, Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma galiisepiicum, Mycoplasma ailigatoris, Mycoplasma mobile, and
Mycoplasma capricolum.
5. The recombinant chimeric AD! of claim 1 , wherein the second microorganism optionally differs from the first microorganism and is selected from the genera Mycoplasma, Clostridium, Bacillus, Borrelia, Enterococcus, Streptococcus,
Lactobacillus, and Giardia.
6. The recombinant chimeric ADI of claim 1 , wherein the second microorganism optionally differs from the first microorganism and is selected from the group consisting of Mycoplasma pneumonia, Mycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis, Streptococcus pyogenes, streptococcus pneumonia, Borrelia burgdorferi, Borrelia afze!ii, Giardia intestinalis, Clostridium perfringens, Bacillus licheniformis, and Enterococcus faecaiis.
7. The recombinant chimeric AD! of claim 1 , wherein the second microorganism optionally differs from the first microorganism and is selected from the group consisting of M. arginini, M. arthritidis, M. hominis, Mycoplasma pneumonia, Mycoplasma phocicerebrale, Mycoplasma orale, Mycoplasma galeae, Mycoplasma phocidae, Mycoplasma columbinum, Mycoplasma iowae, Mycoplasma crocodyli, Mycoplasma fermentans, Mycoplasma penetrans, Mycoplasma gallisepticum, Mycoplasma aliigatoris, Mycoplasma mobile, and Mycoplasma capricoium.
8. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is selected from the group consisting of Mycoplasma gaiiinarum, Mycoplasma iners, and Mycoplasma columbinum and wherein the second microorganism optionally differs from the first microorganism and is selected from the group consisting of Mycoplasma gaiiinarum, Mycoplasma iners, and Mycoplasma columbinum.
9. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is M. arginini and the second microorganism is M. arthritidis.
1 (3. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is M. arginini and the second microorganism is M. hominis.
1 1 . The recombinant chimeric ADI of claim 1 , wherein the first microorganism is M. arthritidis and the second microorganism is M. arginini.
12. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is M. galeae and the second microorganism is M. arthritidis.
13. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is M. gateae and the second microorganism is M. columbinum.
14. The recombinant chimeric ADI of claim 1 , wherein the first microorganism is M. gateae and the second microorganism is M. phocicerebrale.
15. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is M. gateae and the second microorganism is M. phocidae.
16. The recombinant chimeric AD! of ciaim 1 , wherein the first microorganism is M. phocicerebraie and the second microorganism is M. ailigatoris.
17. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is M. phocicerebraie and the second microorganism is M. galeae.
18. The recombinant chimeric AD! of ciaim 1 , wherein the first microorganism is M. phocicerebraie and the second microorganism is M. phocidae.
19. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is M. phocidae and the second microorganism is M, ailigatoris.
20. The recombinant chimeric AD! of ciaim 1 , wherein the first microorganism is M. phocidae and the second microorganism is M, arthritidis.
21. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is M. phocidae and the second microorganism is M, columbinum.
22. The recombinant chimeric AD! of ciaim 1 , wherein the first microorganism is M. phocidae and the second microorganism is M. gateae.
23. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is M. phocidae and the second microorganism is M. phocicerebraie.
24. The recombinant chimeric AD! of ciaim 1 , wherein the first microorganism is M. gallinarum and the second microorganism is M. arthritidis.
25. The recombinant chimeric AD! of ciaim 1 , wherein the first microorganism is M. gallinarum and the second microorganism is M. iners.
26. The recombinant chimeric AD! of claim 1 , wherein the first microorganism is M. iners and the second microorganism is M. columbinum.
27. The recombinant chimeric AD! of daim 1 , wherein the first microorganism is M. iners and the second microorganism is M. gallinarum.
28. The recombinant chimeric AD! of daim 1 , wherein the recombinant chimeric ADI comprises the amino acid sequence set forth in any one of SEQ ID NOs:4-13 or 22-59 or a variant having at ieast 90% identity to any one of SEQ !D NOs:4~13 or 22- 59.
29. The recombinant chimeric ADI of any one of the preceding claims, wherein the recombinant chimeric ADI has been modified to remove at Ieast one pegylation site.
30. The recombinant chimeric AD! of any one of the preceding claims, wherein at Ieast one lysine residue has been modified by an amino acid substitution.
31. The recombinant chimeric AD! of claim 30, wherein at ieast 5 lysine residues have been modified by an amino acid substitution.
32. The recombinant chimeric AD! of claim 30, wherein at ieast 10 lysine residues have been modified by an amino acid substitution.
33. The recombinant chimeric AD! of claim 30, wherein at ieast 15 lysine residues have been modified by an amino acid substitution.
34. The recombinant chime ic AD! of claim 30, wherein at ieast 20 lysine residues have been modified by an amino acid substitution.
35. The recombinant chimeric AD! of claim 30, wherein the recombinant chimeric AD! comprises the amino acid sequence set forth in any one of SEQ ID NOs:10-13.
36. The recombinant chimeric AD! of any one of the preceding claims covalentiy bonded via a biocompatible linker to polyethylene glycol.
37. The recombinant chimeric AD! of claim 30, wherein the arginine deiminase is covalentiy bonded to more than one polyethylene glycol molecule.
38. The recombinant chimeric AD! of claim 30, wherein the arginine deiminase is covalently bonded to about 1 to about 10 polyethylene glycol molecules.
39. The recombinant chimeric AD! of claim 30, wherein the arginine deiminase is covalently bonded to 5±3 PEG molecules.
40. The recombinant chimeric AD! of claim 30, wherein the PEG molecules are straight chain or branch chain PEG molecules.
41. The recombinant chimeric AD! of claim 30, wherein the polyethylene glycol has a total weight average molecular weight of from about 1 ,000 to about 40,000.
42. The recombinant chimeric AD! of claim 30, wherein the polyethylene glycol has a total weight average molecular weight of from about 10,000 to about 30,000.
43. The recombinant chimeric AD! of claim 30, wherein the biocompatible linker comprises a succinyl group, an amide group, an imide group, a carbamate group, an ester group, an epoxy group, a carboxyi group, a hydroxy! group, a carbohydrate, a tyrosine group, a cysteine group, a histidine group, a methylene group, or a combination thereof.
44. The recombinant chimeric AD! of claim 30, wherein the source of the succinyl group is succinimidy! succinate.
45. A polynucleotide encoding the recombinant chimeric AD! of any one of the preceding claims.
46. A vector comprising the polynucleotide of claim 45.
47. An isolated host ceil comprising the vector of claim 48.
48. A composition comprising the recombinant chimeric ADI of any one of the preceding claims and a physiologically acceptable carrier.
49. The composition of claim 48, further comprising an autophagy modulator.
50. The composition of claim 49, wherein the autophagy modulator is selected from the group consisting of chloroquine, 3-methyladenine, hydroxychloroquine, bafiiomycin A1 , 5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid, N8-mercaptopurine riboside, vinblastine, wortmannin, rapamycin, everolimus, metformin, perifosine, resveratrol, and tamoxifen.
51. The composition of claim 48, further comprising a chemotherapeutic agent.
52. The composition of claim 51 , wherein the chemotherapeutic agent is selected from the group consisting of docetaxei, carbopiatin, cyclophosphamide, gemcitabine, cispiatin, sorafenib, sunitinib and everolimus.
53. A method of treating, ameliorating the symptoms of, or inhibiting the progression of a cancer comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 48, thereby treating, ameliorating the symptoms of, or inhibiting the progression of the cancer.
54. The method of claim 53, wherein the cancer is selected from the group consisting of melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, glioma, glioblastoma multiforme, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer.
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